Charge switch nucleotides

ABSTRACT

The present invention provides compounds, methods and systems for sequencing nucleic acid using single molecule detection. Using labeled NPs that exhibit charge-switching behavior, single-molecule DNA sequencing in a microchannel sorting system is realized. In operation, sequencing products are detected enabling real-time sequencing as successive detectable moieties flow through a detection channel. By electrically sorting charged molecules, the cleaved product molecules are detected in isolation without interference from unincorporated NPs and without illuminating the polymerase-DNA complex.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNos. 60/209,896, filed Jun. 7, 2000, and 60/286,238, filed Apr. 24,2001, both the disclosures of which are hereby incorporated by referencein their entirety for all purposes. This application is related to U.S.patent application Ser. No. ______ bearing Attorney Docket Number020031-000820, filed on even date herewith, which is hereby incorporatedby reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

The primary sequences of nucleic acids are crucial for understanding thefunction and control of genes and for applying many of the basictechniques of molecular biology. In fact, rapid DNA sequencing has takenon a more central role after the goal to elucidate the entire humangenome has been achieved. DNA sequencing is an important tool in genomicanalysis as well as other applications, such as genetic identification,forensic analysis, genetic counseling, medical diagnostics, and thelike. With respect to the area of medical diagnostic sequencing,disorders, susceptibilities to disorders, and prognoses of diseaseconditions, can be correlated with the presence of particular DNAsequences, or the degree of variation (or mutation) in DNA sequences, atone or more genetic loci. Examples of such phenomena include humanleukocyte antigen (HLA) typing, cystic fibrosis, tumor progression andheterogeneity, p53 proto-oncogene mutations and ras proto-oncogenemutations (see, Gyllensten et al., PCR Methods and Applications, 1:91-98 (1991); U.S. Pat. No. 5,578,443, issued to Santamaria et al.; andU.S. Pat. No. 5,776,677, issued to Tsui et al.).

Various approaches to DNA sequencing exist. The dideoxy chaintermination method serves as the basis for all currently availableautomated DNA sequencing machines. (see, Sanger et al., Proc. Natl.Acad. Sci., 74: 5463-5467 (1977); Church et al., Science, 240: 185-188(1988); and Hunkapiller et al., Science, 254: 59-67 (1991)). Othermethods include the chemical degradation method, (see, Maxam et al.,Proc. Natl. Acad. Sci., 74: 560-564 (1977), whole-genome approaches(see, Fleischmann et al., Science, 269, 496 (1995)), expressed sequencetag sequencing (see, Velculescu et al., Science, 270, (1995)), arraymethods based on sequencing by hybridization (see, Koster et al., NatureBiotechnology, 14, 1123 (1996)), and single molecule sequencing (SMS)(see, Jett et al., J. Biomol. Struct. Dyn. 7, 301 (1989) and Schecker etal., Proc. SPIE-Int. Soc. Opt. Eng. 2386, 4 (1995)).

PCT Application No. US99/29585, filed Dec. 13, 1999, and incorporatedherein by reference, discloses a single molecule sequencing method on asolid support. The solid support is optionally housed in a flow chamberhaving an inlet and outlet to allow for renewal of reactants that flowpast the immobilized polymerases. The flow chamber can be made ofplastic or glass and should either be open or transparent in the planeviewed by the microscope or optical reader. Electro-osmotic flowrequires a fixed charge on the solid support and a voltage gradient(current) passing between two electrodes placed at opposing ends of thesolid support. The flow chamber can be divided into multiple channelsfor separate sequencing.

Much more recently, PCT Application No. US00/13677, filed May 18, 2000,discloses a method of sequencing a target nucleic acid molecule having aplurality of bases. The temporal order of base additions during thepolymerization reaction is measured on a molecule of nucleic acid. Theactivity of a nucleic acid polymerizing enzyme on the template nucleicacid molecule is thereafter followed in time. The sequence is deduced byidentifying which base is being incorporated into the growingcomplementary strand of the target nucleic acid by the polymerizingenzyme at each step in the sequence of base additions. The steps ofproviding labeled nucleotide analogs, polymerizing the growing nucleicacid strand, and identifying the added nucleotide analog are repeated sothat the nucleic acid strand is further extended and then sequenced.

In addition, U.S. Pat. No. 4,979,824, describes that single moleculedetection can be achieved using flow cytometry wherein flowing samplesare passed through a focused laser with a spatial filter used to definea small volume. Moreover, U.S. Pat. No. 4,793,705 describes a detectionsystem for identifying individual molecules in a flow train of theparticles in a flowcell. The patent further describes methods ofarranging a plurality of lasers, filters and detectors for detectingdifferent fluorescent nucleic acid base-specific labels.

Single molecule detection on solid supports is described in Ishikawa, etal. Jan. J. Apple. Phys. 33:1571-1576. (1994). As described therein,single-molecule detection is accomplished by a laser-inducedfluorescence technique with a position-sensitive photon-countingapparatus involving a photon-counting camera system attached to afluorescence microscope. Laser-induced fluorescence detection of asingle molecule in a capillary for detecting single molecules in aquartz capillary tube has also been described. The selection of lasersis dependent on the label and the quality of light required. Diode,helium neon, argon ion, argon-krypton mixed ion, and Nd:YAG lasers areuseful in this invention (see, Lee et al. (1994) Anal. Chem.,66:4142-4149).

A need currently exists for more effective and efficient compounds,methods, and systems for single molecule detection, especially as theyrelate to single molecule DNA sequencing. These and further needs areprovided by the present invention.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to compounds, methods and systems todetermine and elucidate sequences of nucleic acids. Advantageously, thecompounds, methods and systems of the present invention can be used tosequence nucleic acid rapidly and without the need for amplification orcloning.

In one embodiment, the present invention provides a charge-switchnucleotide phosphate (NP) probe, comprising: an intact NP probe having aterminal phosphate with a fluorophore moiety attached thereto, theintact NP probe having a first molecular charge associated therewith,whereupon cleavage of the terminal phosphate as a phosphate fluorophoremoiety, the phosphate fluorophore moiety carries a second molecularcharge, wherein the difference between the first molecular charge andthe second molecular charge is at least 0.5. Preferably, the differencebetween the first molecular charge and the second molecular charge is atleast 0.5 as calculated in pure water at pH 7.0. In preferred aspects,the charge difference is between about 1 and about 4, and any fractiontherebetween. In certain preferred embodiments, the NP probe has apositive charge, or alternatively, upon cleavage of the terminalphosphate as a phosphate fluorophore moiety, the phosphate fluorophoremoiety carries a positive charge relative to the NP probe.

In a preferred aspect, the NP probe is a nucleotide triphosphate (NTP),and the terminal phosphate is a γ-phosphate with a fluorophore moietyattached thereto. In certain aspects, the NP probe is incorporated intoa growing nucleic acid strand that is complementary to a target nucleicacid, where upon a γ-phosphate with a fluorophore moiety attachedthereto is released as a detectable pyrophosphate moiety.

In one embodiment, the present invention provides an intactcharge-switch nucleotide phosphate (NP) probe, wherein, upon enzymaticcleavage of the intact charge-switch NP probe to produce a phosphatedetectable moiety, the phosphate detectable moiety migrates to anelectrode, and the intact charge-switch NP probe migrates to the otherelectrode.

In another embodiment, the present invention provides a method forseparating a labeled nucleotide phosphate having a detectable moietyfrom a released charged detectable moiety in a sample stream, the methodcomprising: a) immobilizing a complex comprising a nucleic acidpolymerase or a target nucleic acid onto a solid support in a singlemolecule configuration; b) contacting the complex with a sample streamcomprising a target nucleic acid when the polymerase is immobilized, ora polymerase when the target nucleic acid is immobilized, a primernucleic acid which complements a region of the target nucleic acid; anda labeled nucleotide phosphate having a detectable moiety, wherein thedetectable moiety is released as a charged detectable moiety when the NPis incorporated into the primer nucleic acid; and c) applying an energyfield to the sample stream, thereby separating the labeled NP from thecharged detectable moiety.

In certain aspects, the NP is a labeled nucleotide triphosphate (NTP)having a detectable moiety and the detectable moiety is a γ-phosphatewith a fluorophore moiety attached thereto. In a preferred aspect, thecharge of the detectable moiety after release is different than thelabeled nucleotide phosphate (NP) having a detectable moiety attachedthereto.

In another embodiment, the present invention provides a method forsequencing a target nucleic acid comprising: a) immobilizing a complexcomprising a nucleic acid polymerase, or a target nucleic acid onto asolid support in a single molecule configuration; b) contacting thecomplex with a sample stream comprising a target nucleic acid when thepolymerase is immobilized, or a polymerase when the target nucleic acidis immobilized, a primer nucleic acid which complements a region of thetarget nucleic acid of the region to be sequenced; and a labelednucleotide phosphate (NP) having a detectable moiety, wherein thedetectable moiety is released as a charged detectable moiety when the NPis incorporated into the primer nucleic acid wherein the solid supportis disposed in a flowcell having an inlet port and an outlet port; c)applying an energy field to the sample stream; and d) detecting thecharged detectable moiety, thereby sequencing the target nucleic acid.In preferred aspects, the energy field is a first energy field such asan electric field applied in the transverse direction, and a secondenergy filed such as a pressure field applied in the axial direction.The nucleotide phosphate is preferably a nucleotide triphosphate.

Suitable nucleobases include, but are not limited to, adenine, guanine,cytosine, uracil, thymine, deazaadenine and deazaguanosine. In apreferred embodiment, the NPs are charge-switch γ-phosphate labeleddNTP. In one aspect, the polymerase is immobilized and the sample streamcontains a target nucleic acid. In another aspects, the target nucleicacid is immobilized and the sample stream contains polymerase. Inanother aspect, the method includes applying an electric fieldtransverse to the sample stream to sort between a reagent and a product.

In yet another aspect, the present invention provides a system that canbe used to facilitate the contact of fluorescent-labeled nucleotideswith polymerases, and thereafter remove them away (while emittingsignals) from the optical field of view. The system is especiallybeneficial in single-molecule sequencing schemes to facilitatedetection. As such, the present invention provides a microfabricatedflowcell system for single-molecule detection, comprising: a) a flowcellhaving an inlet port and an outlet port wherein a sample stream having adetectable analyte flows therethrough; b) an energy field source appliedto the sample stream; and c) a detector for detecting the analyte.

In certain aspects, the system comprises two energy fields, one axial tothe sample stream and the other energy field applied in the transversedirection. Preferably, the applied fields are electric fields, pressurefields and combinations thereof. The fields are variable, thuspermitting control of the motion of the nucleotides and (afterincorporation) the phosphate detectable moiety (e.g.,fluorescent-labeled phosphate).

In certain embodiments, the flowcell has multiple inlet ports andmultiple outlet ports wherein a sample stream having detectable analytesflow therethrough. In addition to a first energy field and a secondenergy field, in certain aspects, the flowcell of the present inventioncomprises an array of energy fields disposed throughout the flowcellarrangement and an array of immobilized polymerases, target nucleicacids and combinations thereof in single molecule configuration. Thisarrangement can be used to analyze a plurality of nucleic acids in asingle flowcell device.

Numerous benefits and advantages are achieved by way of the presentinvention over conventional compounds, methods and systems. For example,the charge-switch nucleotide phosphates allow separation of the cleavedterminal phosphate (e.g., pyrophosphate) from the intact nucleotidephosphate probe reagents. This characteristic is useful forsingle-molecule DNA sequencing in a microchannel sorting system with anenergy field. Using 4 different NTPs each labeled with a unique dye,real-time DNA sequencing is possible by detecting the releasedpyrophosphate having different labels. By electrically sortingdifferently-charged molecules in this manner, the cleaved PPi-Dyemolecules are detected in isolation without interference fromunincorporated NTPs and without illuminating the polymerase-DNA complex.

With respect to the flowcell, the energy fields can be varied inaccordance with the charge on a molecule to increase the probability ofsignal detection. Moreover, the flowcell of the present system increasesthe signal-to-noise ratio (S/N) of the detectable moiety. By increasingthe S/N, a lower detection limit is possible.

These and other objects and advantages will become more apparent whenread with the accompanying detailed description and drawings thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a model compound of the present invention;

FIG. 2 tabulates various charges of charge-switch nucleotides accordingto the present invention. In the ideal condition, the charged groupsattached to the nucleobase, sugar or on the label “F” are assumed to bein fully charged form. In the pure water condition, the effect ofhydrogen ions on the net charge of the phosphate groups is calculatedusing equilibrium constants given by Frey and Stuhr (1972), Journal ofAmerican Chemical Society, 94:8818. Hydrogen ions confer a time-averagedpartial positive charge preferentially to the PPi-F group as compared tothe NP Probe due to the presence of the secondary ionization phosphateoxygen present only on the PPi-F group;

FIG. 3 (A-F) illustrates schematically equilibrium calculations showingthe effect of Mg⁺⁻ on the time-averaged electric charge on the “ligands”N-PPP-F and PP-F (N=nucleotide, PPP=triphosphate, PP=pyrophosphate,F=γ-label). Binding to the ions H⁺ and Mg⁺⁺ are considered. The fractionof ligand bound to an ion, fracBound, is given asfracBound=[ion]/([ion]+K), where K is the ion concentration givingfracBound=50% (i.e., the association or dissociation constant). Thefractions of N-PPP-F and PP-F in protonated form were calculatedaccording to the above eqn. Then, the fraction bound to Mg⁺⁺ wascalculated for both the protonated and unprotonated forms of N-PPP-F andPP-F. The average charge was then calculated by multiplying the fractionof each form by its respective charge and adding all of the forms of themolecule. Results are plotted as a function of Mg⁺⁺ concentration (0-25mM). Charges on N and F were modeled as pH-independent quaternary salts(+) or carboxylates (−) PANEL A N(0) F(0), PANEL B N(0) F(2), PANEL CN10) F(0), PANEL D N(2) F(0), PANEL E N(−1) F(2), PANEL E N(−2) F(3);

FIG. 4 illustrates a compound of the present invention (dTTP-BQS-BTR);

FIG. 5 illustrates a schematic of equilibrium calculations of thepresent invention (see, Example I);

FIG. 6 (A-F) PANEL A compounds of the present invention; PANEL Bcompounds of the present invention; PANEL C compounds of the presentinvention; PANEL D compounds of the present invention; PANEL E variouslinker embodiments used in compounds of the present invention; and PANELF various linker embodiments used in compounds of the present invention;

FIG. 7 illustrates a schematic of an embodiment of microfabricatedflowcell of the present invention;

FIG. 8 illustrates a schematic of a method embodiment of the presentinvention;

FIG. 9 illustrates a schematic of an embodiment of microfabricatedflowcell of the present invention;

FIG. 10 illustrates a schematic of an embodiment of microfabricatedflowcell of the present invention;

FIG. 11 PANEL A nucleotide electrophoretic velocities are plotted as afunction of Mg⁺⁺ concentration. Panel B effect of Mg⁺⁺ onelectrophoretic migration of the compound in FIG. 4 in agarose gelscontaining the indicated amounts of Mg⁺⁺;

FIG. 12 illustrates a synthetic scheme of an embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS I.Definitions

The term “charge-switch nucleotide” as used herein refers to a labelednucleotide phosphate (e.g., γ-NP-Dye) that upon release or cleavage of aphosphate detectable moiety (e.g., PPi-Dye) has a different net chargeassociated with the cleavage product compared to the intact nucleotidephosphate probe (e.g., γ-NP-Dye). In certain preferred aspects, theattachment of the dye to the PPi is via a nitrogen in lieu of an oxygen.

Preferably, the charge difference between the intact γ-NP-Dye and thePPi-Dye is at least 0.5, and more preferably about 1 to about 4 (e.g.,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, and 4.0).

The terms “PPi-Dye” or “PP-F” and the like, refer to the pyrophosphatecleavage product from an intact charge-switch nucleotide (NTP). If anucleotide diphosphate is used, the cleavage product will be a “P-Dye”or “P-F”.

The phrase “phosphate detectable moiety” refers to a detectable cleavageproduct from a NP probe of the present invention. Examples include, butare not limited to, PPi-Dye, PP-F, P-Dye, a phosphate fluorophoremoiety, a terminal phosphate fluorophore moiety, a detectable moiety,charged groups, electrically active groups, detectable groups, reportergroups, combinations thereof, and the like.

The term “heterogeneous” assay as used herein refers to an assay methodwherein at least one of the reactants in the assay mixture is attachedto a solid phase, such as a solid support.

The term “oligonucleotide” as used herein includes linear oligomers ofnucleotides or analogs thereof, including deoxyribonucleosides,ribonucleosides, and the like. Usually, oligonucleotides range in sizefrom a few monomeric units, e.g. 3-4, to several hundreds of monomericunits. Whenever an oligonucleotide is represented by a sequence ofletters, such as “ATGCCTG,” it will be understood that the nucleotidesare in 5′-3′ order from left to right and that “A” denotesdeoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine,and “T” denotes thymidine, unless otherwise noted.

The term “nucleoside” as used herein refers to a compound consisting ofa purine, deazapurine, or pyrimidine nucleoside base, e.g., adenine,guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, andthe like, linked to a pentose at the 1′ position, including 2′-deoxy and2′-hydroxyl forms, e.g., as described in Kornberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992).

The term “nucleotide” as used herein refers to a phosphate ester of anucleoside, e.g., mono, di and triphosphate esters, wherein the mostcommon site of esterification is the hydroxyl group attached to the C-5position of the pentose. Nucleosides also include, but are not limitedto, synthetic nucleosides having modified base moieties and/or modifiedsugar moieties, e.g. described generally by Scheit, Nucleotide Analogs(John Wiley, N.Y., 1980). Suitable NTPs include both naturally occurringand synthetic nucleotide triphosphates, and are not limited to, ATP,dATP, CTP, dCTP, GTP, dGTP, TTP, dTTP, UTP and dUTP. Preferably, thenucleotide triphosphates used in the methods of the present inventionare selected from the group of dATP, dCTP, dGTP, dTTP, dUTP and mixturesthereof.

The term “primer” refers to a linear oligonucleotide, which specificallyanneals to a unique polynucleotide sequence and allows for synthesis ofthe complement of the polynucleotide sequence. In certain aspects, aprimer is covalently attached to the template as a hairpin.

The phrase “sequence determination” or “determining a nucleotidesequence” in reference to polynucleotides includes determination ofpartial as well as full sequence information of the polynucleotide. Thatis, the term includes sequence comparisons, fingerprinting, and likelevels of information about a target polynucleotide, or oligonucleotide,as well as the express identification and ordering of nucleosides,usually each nucleoside, in a target polynucleotide. The term alsoincludes the determination of the identification, ordering, andlocations of one, two, or three of the four types of nucleotides withina target polynucleotide.

The term “solid-support” refers to a material in the solid-phase thatinteracts with reagents in the liquid phase by heterogeneous reactions.Solid-supports can be derivatized with proteins such as enzymes,peptides, oligonucleotides and polynucleotides by covalent ornon-covalent bonding through one or more attachment sites, thereby“immobilizing” the protein or nucleic acid to the solid-support.

The phrase “target nucleic acid” or “target polynucleotide” refers to anucleic acid or polynucleotide whose sequence identity or ordering orlocation of nucleosides is to be determined using methods describedherein.

The phrase “terminal phosphate oxygen” refers to the secondaryionization oxygen atom (pK ˜6.5) attached to the terminal phosphate atomin a nucleotide phosphate probe.

The phrase “internal phosphate oxygen” refers to the primary ionizationoxygen atoms (pK ˜2) in a nucleotide phosphate probe. An NTP has 3internal phosphate oxygens (one each on the α, β, and γ-phosphates) plus1 terminal phosphate oxygen (on the γ-phosphate).

The phrase “single molecule configuration” refers to the ability of thecompounds, methods and systems of the present invention to measuresingle molecular events, such as an array of molecules on a solidsupport wherein members of the array are present as individual moleculeslocated in a defined location. The members can be the same or different.

II. Compounds

In one embodiment, the present invention provides a charge-switchnucleotide phosphate (NP) probe. The NP probe has a terminal phosphatewith a fluorophore moiety attached thereto. The NP probe can be anucleotide diphosphate or nucleotide triphosphate. Preferably, thecharge-switch NP probe is a nucleotide triphosphate. In certainpreferred aspects, the nucleoside moiety is modified with adducts toconfer positive or negative charge. As explained in detail below,modification can occur at the base, the sugar, the phosphate group,linkers and combinations thereof. Advantageously, by electricallysorting molecules having different charges relative to each other, suchas by separating an intact charge switch nucleotide from its cleavedPPi-Dye, the cleaved PPi-Dye (PPi-F) molecules are detected in isolationwithout interference from unincorporated NP probes (e.g., γ-NP-Dye).

In certain embodiments, the incorporation of an NP probe in the growingcomplementary strand of nucleic acid results in release of a phosphatedetectable moiety. In a preferred embodiment, the detectable moiety is aγ-phosphate label that is cleaved from γ-labeled dNTPs by a polymerase.In an especially preferred embodiment, γ-labeled-dNTPs having a cationicγ-label exhibit charge-switching behavior, wherein the electric chargeof the intact triphosphate (γ-NTP-Dye) is negative while the releasedPPi-Dye is positive. Thus, the release of the PPi-Dye results in acleavage-dependent charge alteration to the PPi-fluorophore moiety. Incertain aspects, cleavage of pyrophosphate from the nucleoside subtractscharges associated with the nucleoside. These charge changes can beeither positive or negative. In certain aspects, the cleavage of thePPi-Dye adds a positive charge to the PPi-Dye moiety by generating aterminal phosphate oxygen, as a terminal phosphate-oxygen binds mono ordivalent cations (e.g., Mg⁺⁺, Mn⁺⁺, K⁺, Na⁺ and the like) as counterions, better than an internal phosphate-oxygen.

In certain aspects, the charge-switch NP probes of the present inventionhave a net positive charge. For example, the base can have an amineattached thereto and this amine can be protonated. Upon cleavage of thebase-cation, the PPi-Dye becomes more negative. Conversely, cleavage ofa negative-base NP (e.g., a base with a carboxylate, sulfonate, and thelike attached thereto) makes the PPi-Dye more positively charged.Cleavage of a neutral-base NTP (natural structure), will have nocontribution to the PPi-Dye other than generation of a terminalphosphate oxygen.

A. Charge State

The charge state of the NP probe as well as the released terminalphosphate (e.g., pyrophosphate) are parameters of the compounds of thepresent invention. Those of skill in the art will appreciate the variousparameters making-up or contributing to the charge on the γ-NP-Dye andthe terminal phosphate-Dye (e.g., PPi-Dye moiety). In certain aspects, acharge-switch nucleotide phosphate (NP) probe comprises an intact NPprobe having a terminal phosphate with a fluorophore moiety attachedthereto. The intact NP probe has a first molecular charge associatedtherewith; and whereupon cleavage of the terminal phosphate such ascleavage of a pyrophosphate fluorophore moiety, the pyrophosphatefluorophore moiety carries a second molecular charge. The firstmolecular charge is different than the second molecular charge by atleast 0.4 as calculated under ionic conditions obtained in pure water,at about pH 7 (see, FIG. 2). The charge difference between the intact NPprobe is more preferably between about 1 and about 4, and any fractionof the integers 1, 2, and 3

The charge state of the either the γ-NP-Dye or terminal phosphate-Dye(e.g., PPi-Dye) or both can be determined for any ionic condition bycalculating the i) charge on the base; the ii) charge on the fluorophoreor linker; and iii) the buffer cation composition and concentration(see, Example I).

In general, the net electric charge on a nucleotide phosphate such as adNTP, is governed by the base ring nitrogens and by the threephosphates. At a pH from about 6.5 to about 8.5, the bases are mostlyuncharged (nitrogen pK of 3-4 and 9.5-10). The primary ionization ofeach ionizable oxygen atom on each phosphate (pK ˜2) contributes onefull negative charge. The secondary ionization specific to the phosphateoxygen (pK ˜6.5) contributes a time-averaged charge of −0.9 at pH 7.5 sothe total charge on the dNTP is −3.9.

FIG. 1 illustrates a representative compound of the present inventionshowing an intact γ-NP-Dye and the released pyrophosphate having adetectable moiety. As shown therein, in certain aspects, the nucleobasecarries a cationic adduct and the terminal oxygen is replaced by anitrogen and a label moiety in a γ-dNTP, thus, the secondary ionizationis eliminated and at pH 7 (H₂O), the charge on a γ-dNTP is −2.0 (for aneutral γ-label). After cleavage from the nucleotide, the charge on thePPi-Dye is −2.74, because it has lost the positive charge (+1) of thenucleobase, but has gained back a partial positive charge (+0.26) due tohydrogen ion equilibration with the terminal phosphate oxygen (pK 6.4secondary ionization of substituted diphosphates).

FIG. 2 is a look-up table showing various embodiments and chargesassociated with the nucleobase and dye and their respective net chargesunder ideal conditions (without associated counter ions or buffers;charged adducts fully charged) or in pure water (last column only).Entry 32 illustrates the preceding example. As tabulated therein, forideal conditions, the nucleobase has a charge of 1, the dye has a chargeof 0 and therefore a net charge of −2 is associated with the γ-NP-Dyeand a charge of −3 for the PPi-Dye, giving a charge difference of −1.The charge difference is slightly less (−0.74; last column) in purewater at pH 7, however, because the terminal phosphate oxygen of PPi-Fassociates more readily with hydrogen ions. As shown therein, variouselectric charges placed on the nucleobase and the dye will havedifferent effects on the dye upon incorporation of the nucleobase into agrowing nucleic acid. The charge difference under ideal conditions isequal to the sum of the opposite of the charge on the nucleobase moietyand the terminal phosphate dye moiety, as the nucleobase is separatedfrom the dye when the nucleobase is incorporated into DNA. The chargedifference in pure water (last column) takes into account hydrogen ionequilibrium binding.

In certain other embodiments, the charge-switch NP probes of the presentinvention have various counter ions associated with them (e.g., Mg⁺⁺ orother cations). For example, Mg⁺⁺ binds to phosphate groups in a varietyof coordination isomers that rapidly equilibrate at 10³ to 10⁵ sec⁻¹.Mg⁺⁺ ions, like protons, bind more tightly to terminal phosphates thanto “internal” phosphates, meaning that a PPi-Dye moiety acquires morepositive charge from the counter ions than a γ-dNTP-Dye. In operation,this difference is utilized to sort or separate the released PPi-Dyefrom the intact γ-NTP-Dyes in for example, a microchannel system usingthe compounds, methods and systems of the present invention.

As explained in more detail below, the magnitude of a charge-switch canbe enhanced by attaching positive or negative charged groups to thenucleoside (normally neutral at pH 7.5). The range of the charge-switchcan be set by attaching charged groups to the γ-phosphate label, eitheron the fluorophore and/or linker, such that both the NP probe and thePPi-F are negatively charged, or both are positively charged, or one isnegative while the other is positive. All such combinations andpermutations are encompassed by the present invention. Thereafter, whenthe base is incorporated into DNA, the charged group is separated fromthe PPi-F to enhance the “natural” counter ion (e.g., Mg⁺⁺) dependentcharge effect.

There are 10 charge-switch modes that can be exploited for sorting (negto weak neg, neg to strong negative, neg to zero, neg to pos, zero toneg, zero to pos, pos to neg, pos to zero, weak to strong pos and strongto weak pos). The two “bipolar” modes i.e., negative to positive,positive to negative are preferred for electrosorting, although theother modes can also be used under appropriate microfluidic conditions.Other preferred compounds from FIG. 2 are set forth in Table 1.

TABLE 1 Compound Charges 20 N(−1) F(+2) 13 N(−2) F(+2) 12 N(−2) F(+1) 40N(+2) F(+1) 39 N(+2) F(0) 19 N(−1) F(+1) 27 N(0) F(+2) 33 N(+1) F(+1) 26N(0) F(+1) 41 N(+2) F(+2)

In order to obtain a bipolar mode, the γ-dNTP is “poised” with respectto charge so that the charge switch “passes through” neutral. FIG. 3(A-F) illustrate how the counter ion concentration (e.g., Mg⁺⁺ ion)affects the charge of a generic γ-nucleotide (N—PPP-F) and a cleavageproduct (PP-F). Six different charge configurations “N(b) F(g)” areshown (A-F) wherein b and g are the charge on the nucleoside or γ-label,respectively. The charged groups (having different pK's) can be forexample, primary or quaternary amines which add positive charge (+), ora carboxylic acid, which adds negative charge (−) and the like. With noadded groups N(0) F(0) (Panel A), the maximum charge switch at pH 8(Δq=+1) occurs at about 2 mM Mg⁺⁺, with the change being in negativerange (−2.5 to −1.5). By adding a charge of (+2) to the γ-label (PanelB), the same switch magnitude is obtained (Δq=+1), except now the shiftis a bipolar mode wherein the γ-dNTP-F and PPi-F are oppositely charged(−0.5 to +0.5). Other configurations in FIG. 3 (C-F) show how the chargeswitch magnitude can be further increased (to facilitate electrosorting)by adding various charges to the nucleobase and/or γ-label. As isapparent from FIG. 3, the charge difference (Δq) can occur in negativerange, positive range, negative to positive range or positive tonegative range.

As exemplified in FIG. 4, the charge difference between the intact NPprobes and the detectable moieties can be introduced via a chargedmoiety fixed to the γ-label such that, the γ-NTP-Dye is net negative,while the PPi-Dye is net positive. As shown therein, the electroneutraldye BODIPY®TR is conjugated to dTTP using a linker having a charge of+2. This nucleotide can be incorporated into DNA by a polymerase, withthe release of phosphate, thus the PPi-Linker-Dye moiety acquires a morepositive charge than the intact γ-NTP-Dye.

Using the equations set forth in Example I below, and with reference toFIG. 5, it is possible to calculate the net charge on the γ-NP-Dye andthe released terminal phosphate (e.g., PPi-Dye) in the presence andabsence of a metal counter ion. In certain instances, equilibriumassociation of cations to the compounds of the present invention willadd about 1 positive charge to Δq, depending on the cation composition,concentration and pH (see, FIG. 3).

The determination of charge on each moiety can be carried out using theequilibrium calculations in Example I below and as illustrated in FIG.5. Using the equilibrium equations, Example I sets forth the chargedetermination of the compound in FIG. 4.

FIG. 6 (A-D) illustrates various charge-switch nucleotides of thepresent invention. These compounds are merely an illustration and shouldnot limit the scope of the claims herein. One of ordinary skill in theart will recognize other variations, modifications, and alternatives.

In certain aspects, the present invention provides a charge-switchnucleotide phosphate (NP) probe. The NP probe has a terminal phosphatewith a fluorophore moiety attached thereto, wherein the intact NP probehas a first molecular charge associated therewith, and upon cleavage ofthe fluorophore moiety having a phosphate or pyrophosphate groupappended thereto, the P-F or PPi-F has a second charge. The first chargeand second charge are different. Formula I provides charge-switchnucleotide phosphate probes of the present invention:

In Formula I, B is a nucleobase including, but not limited to, naturallyoccurring or synthetic purine or pyrimidine heterocyclic bases,including but not limited to adenine, guanine, cytosine, thymine,uracil, 5-methylcytosine, hypoxanthine or 2-aminoadenine. Other suchheterocyclic bases include 2-methylpurine, 2,6-diaminopurine,6-mercaptopurine, 2,6-dimercaptopurine, 2-amino-6-mercaptopurine,5-methylcytosine, 4-amino-2-mercaptopyrimidine, 2,4-dimercaptopyrimidineand 5-fluorocytosine. Representative heterocyclic bases are disclosed inU.S. Pat. No. 3,687,808 (Merigan, et al.), which is incorporated hereinby reference.

In certain preferred aspects, B comprises a charged moiety. Thesecharged base-moieties can be positively or negatively charged. Using acharged base-moiety, it is possible to impart additional charge onto thebase or the intact 7-dNTP-F. Suitable charged base linking groups canappend carboxylic acid group, sulfonic acid group, and the like.

R¹ in Formula I is a hydrogen, a hydroxyl group or charged group e.g.,L-SO₃ ⁻, L-NH₃ ⁺, L-CO₂ ⁻ and the like; wherein L is a linker.

R² in Formula I is a hydrogen, or charged group e.g., L-SO₃ ⁻, L-NH₃ ⁺,L-CO₂ ⁻ and the like; wherein L is a linker.

In Formula I, X is a heteroatom such as nitrogen, oxygen, and sulfur.Preferably, X is nitrogen. As the NP probes of the present invention canbe tetraphosphates, triphosphates or diphosphates, the index “y” inFormula I, can be 0, 1 or 3.

In Formula I, F is a fluorophore or dye. In certain preferred aspects, Fcomprises a charged label linker group. Using the charged label linkinggroup, it possible to impart additional charge onto the fluorophoremoiety (i.e., the cleaved PPi-F or P-F). Suitable charged label-linkinggroups can append quaternary nitrogens and the like. The compounds ofFormula I can have counter ions associated therewith. These counter ionsinclude mono and divalent metal ions including, but are not limited to,Mg⁺⁺, Mn⁺⁺, K⁺ and Na⁺. Those of skill in the art will know ofadditional counter ions suitable for use in the present invention. FIGS.6(A-D) set forth preferred compounds of the present invention.

B. Labels

Many dyes or labels are suitable for charge-switch nucleotide phosphatesof the present invention. In fact, there is a great deal of practicalguidance available in the literature for providing an exhaustive list offluorescent and chromogenic molecules and their relevant opticalproperties (see, for example, Berlman, Handbook of Fluorescence Spectraof Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971);Griffiths, Colour and Constitution of Organic Molecules (Academic Press,New York, 1976); Bishop, Ed., Indicators (Pergamon Press, Oxford, 1972);Haugland, Handbook of Fluorescent Probes and Research Chemicals(Molecular Probes, Eugene, 1992) Pringsheim, Fluorescence andPhosphorescence (Interscience Publishers, New York, 1949); and the like.Further, there is extensive guidance in the literature for derivatizingfluorophore molecules for covalent attachment via common reactive groupsthat can be added to a nucleotide, as exemplified by the followingreferences: U.S. Pat. No. 3,996,345; Khanna et al., and U.S. Pat. No.4,351,760.

In certain preferred aspects, suitable dyes include, but are not limitedto, coumarin dyes, xanthene dyes, resorufins, cyanine dyes,difluoroboradiazaindacene dyes (BODIPY), ALEXA dyes, indoles, bimanes,isoindoles, dansyl dyes, naphthalimides, phthalimides, xanthenes,lanthanide dyes, rhodamines and fluoresceins. In certain embodiments,certain visible and near IR dyes are known to be sufficientlyfluorescent and photostable to be detected as single molecules. In thisaspect the visible dye, BODIPY R6G (525/545), and a larger dye, LI-COR'snear-infrared dye, IRD-38 (780/810) can be detected with single-moleculesensitivity and are used to practice the present invention.

In certain preferred aspects, suitable dyes include, but are not limitedto, fluorescein, 5-carboxyfluorescein (FAM), rhodamine,5-(2′-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS),anthranilamide, coumarin, terbium chelate derivatives, Reactive Red 4,BODIPY dyes and cyanine dyes.

In certain aspects, the phosphate detectable moiety is a charged group.As explained below, Schemes 1-6 in FIG. 6 E sets forth aliphatic linkersfor γ-phosphate conjugation. In certain aspects, the linkers in Schemes1-6 can be used without further attachment of a label such as afluorophore. The linkers themselves can be used as the phosphatedetectable moieties.

C. Linkers to the Label

There are many linking moieties and methodologies for attachingfluorophore moieties to nucleotides, as exemplified by the followingreferences: Eckstein, editor, Oligonucleotides and Analogues. APractical Approach (IRL Press, Oxford, 1991); Zuckerman et al., NucleicAcids Research, 15: 5305-5321 (1987) (3′ thiol group onoligonucleotide); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′ sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′ phosphoamino groupvia Aminolink™ II available from Applied Biosystems, Foster City,Calif.) Stabinsky, U.S. Pat. No. 4,739,044 (3′ aminoalkylphosphorylgroup); AP3 Labeling Technology (U.S. Pat. Nos. 5,047,519 and 5,151,507,assigned to E.I. DuPont de Nemours & Co); Agrawal et al., TetrahedronLetters, 31: 1543-1546 (1990) (attachment via phosphoramidate linkages);Sproat et al., Nucleic Acids Research, 15: 4837 (1987) (5′ mercaptogroup); Nelson et al., Nucleic Acids Research, 17: 7187-7194 (1989) (3′amino group); and the like.

In certain aspects, the fluorophore moiety is a fluorescent organic dyederivatized for attachment to a γ-phosphate directly or via a linker. Ingeneral, nucleotide labeling can be accomplished using any of a largenumber of known nucleotide labeling techniques using known linkages,linking groups, and associated complementary functionalities. Thelinkage linking the fluorophore to the phosphate should be compatiblewith relevant polymerases.

In one embodiment, the linker is an alkylene group, such as a methyleneor ethylene group. In this embodiment, the fluorophore linker is analkylene group having between about 1 to about 50 carbon atoms,preferably about 10 to 30 carbon atoms and more preferably, about 15 toabout 25 carbon atoms, optionally interrupted by heteroatom(s). Incertain aspects, the linker has at least 1 positive or negative chargeassociated therewith.

In certain other embodiments, various charged linkers can also be used.Schemes 1-6 in FIG. 6 E sets forth aliphatic linkers for γ-phosphateconjugation.

As shown therein, Scheme 1 sets forth a MQS(+) (monoquaternary salt)linker generated using a phthaliamide protecting group. The MQS isthereafter used as a reagent in Schemes 3 and 4. Scheme 2 sets forth aBQS(++) (bisquaternary salt) linker. Scheme 3 sets forth a TQS(+++)(triquaternary salt) linker, which is made by combining one MQS unitwith one BQS unit using appropriate stoichiometry. The phthaliamideprotecting group is removed when necessary in 1M NaOH for 2 h. Inaddition, Scheme 4 sets forth a TetQS(++++) (tetraquaternary salt)linker made by combining two MQS units with one BQS unit as shown.Scheme 5 sets forth protection of the aminoally amino group of AA-dUTP,and Scheme 6 sets forth the chemistry to couple the BQS linker to dTTP.The product is purified by HPLC and reacted with the succinimide esterof BodipyTR.

In still other embodiments, FIG. 6F sets forth peptide moieties forlinking the fluorophore to the terminal phosphate. Preferably, thepeptide is between 2 and 15 amino acids in length. Scheme 7 shows thecoupling of 3 lysines (KKK) through their ε-amines so that each residueprovides 7 atoms to the linker. The three lysines together form alargely-aliphatic linker 21 atoms long, about the same size as the BQSlinker. Both the C and N-termini of the peptide are blocked by amidationor acylation. A reversible protecting group is required to achievedirectional coupling. Using a protecting group having the sequence RPTL(C—N direction), it is possible to cleave the peptide linker veryspecifically by thrombin on the C-terminal side of the arginine (Harriset al., Proc Nat Acad Sci USA, 97:7754-7759 (2000)). In addition, Scheme8 shows the peptides of Scheme 7 being coupled directionally to the γ-Pof dNTPs. Additional linkers suitable for use in the present inventionwill be apparent to those of skill in the art.

D. Charged Moieties on the Base

In certain aspects, the base has a charged moiety appended thereto toincrease or decrease molecular charge. In general, attaching one or morenucleotide charged moieties can be accomplished using any of a largenumber of known nucleotide labeling techniques using known linkages,linking groups, and associated complementary functionalities.Preferably, the linkage attaching the charged moiety and nucleotideshould be compatible with relevant polymerases.

Preferably, the charged moieties are covalently linked to the 5-carbonof pyrimidine bases and to the 7-carbon of 7-deazapurine bases. Severalsuitable base labeling procedures have been reported that can be usedwith the present invention, e.g. Gibson et al., Nucleic Acids Research,15: 6455-6467 (1987); Gebeyehu et al., Nucleic Acids Research, 15:4513-4535 (1987); Haralambidis et al., Nucleic Acids Research, 15:4856-4876 (1987); Nelson et al., Nucleosides and Nucleotides, 5(3)233-241 (1986); Bergstrom, et al., JACS, 111, 374-375 (1989); U.S. Pat.Nos. 4,855,225, 5,231,191, and 5,449,767, each of which is incorporatedherein by reference. Preferably, the linkages are acetylenic amido oralkenic amido linkages, the linkage between the charged moiety and thenucleotide base being formed by reacting an activatedN-hydroxysuccinimide (NHS) ester of the charged moiety with analkynylamino- or alkenylamino-derivatized base of a nucleotide.

The synthesis of alkynylamino-derivatized nucleosides is taught by Hobbset al. in European Patent Application No. 87305844.0; U.S. Pat. Nos.5,047,519 and 5,151,507, assigned to E.I. DuPont de Nemours & Co; andHobbs et al., J. Org. Chem., 54: 3420 (1989), which are incorporatedherein by reference. As taught therein, the alkynylamino-derivatizednucleotides are formed by placing the appropriate halodeoxynucleoside(usually 5-iodopyrimidine and 7-iodo-7-deazapurine deoxynucleosides andCu(I) in a flask, flushing with argon to remove air, adding dry DMF,followed by addition of an alkynylamine, triethyl-amine and Pd(0). Thereaction mixture can be stirred for several hours, or until thin layerchromatography indicates consumption of the halodeoxynucleoside.

As taught in U.S. Pat. No. 5,047,519, which issued to Hobbs et al. onSep. 10, 1991, the alkynylamino linkers have the structure:

Nuc-C═C—R¹—NR²R³

wherein R¹ is a substituted or unsubstituted diradical moiety of 1-20atoms. Nuc is a purine or pyrimidine base. R¹ can be straight-chainedalkylene, C₁-C₂₀, optionally containing within the chain double bonds,triple bonds, aryl groups or heteroatoms such as N, O or S. Theheteroatoms can be part of such functional groups as ethers, thioethers,esters, amines or amides. Preferably, R¹ is straight-chained alkylene,C₁-C₂₀; most preferably R¹ is CH₂. Substituents on R¹ can include C₁-C₆alkyl, aryl, ester, ether, amine, amide or chloro groups. R² and R³ areindependently H, alkyl, C₁-C₄, or a protecting group such as acyl,alkoxycarbonyl, a charged moiety or sulfonyl. Preferably R² is H, and R³is a charged moiety. The alkynylamino linker is preferably attached tothe 5-position of the pyrimidine nucleotides and the 7 position of thepurine nucleotides.

In still other embodiments, FIG. 6F sets forth methods for carboxylatingthe aminoally group of AA-dUTP using succinic anhydride (−1) or1,2,4-benzenetricarboxylic anhydride (−2). This provides negativelycharged bases to test the high-magnitude charge-switch configurations.In addition, Scheme 10 shows peptide linkers are used to synthesize thecarboxylated γ-dUTPs mentioned in Scheme 9.

In yet another aspect, the charge group is attached to the sugar.Suitable charged groups and their syntheses are disclosed in U.S. Pat.No. 6,191,266 (incorporated herein by reference). The charged groups canbe at C-2 or C-3 or combinations thereof.

E. Assay to Assess Charge

Those of skill in the art will readily recognize that various assays areeasily implemented to assess the charge of the intact nucleotidephosphate and the cleaved pyrophosphate carrying a label. The followingassay is just one of many available assays to calculate and assess thenet charge on the γ-NP-Dye and the released PPi-F or P-F moiety.

In certain instances, the assay set forth in Example VII is used to testfor a change in the electric charge associated with a dye attached tothe terminal phosphate of a nucleotide. In one embodiment, the chargeswitch is caused by cleavage of a phosphodiester bond that links the dyeto the nucleotide. In one example, cleavage is catalyzed by snake venomphosphodiesterase. It will be appreciated by those of skill in the artthat other enzymes, such as a DNA polymerase listed herein, can also beused to demonstrate charge switching.

As such, in another embodiment, the present invention provides a methodfor identifying an intact charge-switch nucleotide phosphate (NP) probe,comprising: a) contacting a sample comprising the intact charge-switchNP probe with an enzyme to produce a phosphate detectable moiety; and b)applying an electric field to the sample, wherein the phosphatedetectable moiety migrates to an electrode differently than the intactcharge-switch NP probe.

III. Methods

The charge-switch nucleotide phosphate probes of the present inventioncan be used in a variety of methods and systems such as methods andsystems for sequencing nucleic acid. As described above, in certainaspects, the γ-label is cleaved from γ-dNTPs by various polymerases. Inthis reaction, the phosphate ester bond between the α and β phosphatesof the incorporated nucleotide is cleaved by the DNA polymerase, and theβ-γ-diphosphate (pyrophosphate) is released in solution. As used herein,the term pyrophosphate also includes substitution of any of the oxygenatoms of the pyrophosphate group with a nitrogen or a sulfur atom orcombinations thereof to generate thiopyrophosphate, dithiopyrophosphate,and the like. Separating the unincorporated γ-NP-Dyes from the PPi-Dyeis facilitated when the unincorporated γ-NP-Dyes has a net charge thatis different than the released PPi-Dye. For example, a cationic PPi-Dyeand a negative intact γ-NP-Dyes (e.g., triphosphate) exhibit chargeswitching. This characteristic is useful for single-molecule DNAsequencing in a microchannel sorting system for example, where apolymerase-DNA complex is immobilized just upstream from a channelintersection.

A. Separating, Sorting and Sequencing

FIG. 7 is a schematic of a fabricated flowcell system 70 of the presentinvention. This diagram is merely an illustration and should not limitthe scope of the claims herein. One of ordinary skill in the art willrecognize other variations, modifications, and alternatives.

An electric field 71A and 71B at the intersection drives intactγ-dNTP-Dyes into a first microchannel toward the anode 71A, whilePPi-Dye molecules are driven toward the cathode 71B into a secondchannel where they are detected with a detector 74. In operation, eachof the 4 dNTPs is labeled with a different dye, enabling real-timesequencing as successive PPi-γ-Dye molecules flow through the detectionchannel 75. By electrically sorting differently-charged molecules inthis manner, the cleaved PPi-Dye molecules 76 are detected in isolationwithout interference from unincorporated γ-dNTP-Dyes 77 and withoutilluminating the polymerase-DNA complex 79.

In certain aspects, a change in charge sign (e.g., from −1 on theγ-dNTP-Dye to +1 on the PPi-Dye) is utilized to separate the γ-dNTP-Dyefrom the PPi-Dye. In certain aspects, the γ-dNTP-Dye flows across apolymerase located just upstream from a transverse channel. Theγ-dNTP-Dye is hydrolyzed by a polymerase and the liberated PPi-Dyediffuses into the medium and moves towards the transverse channel. Atransverse electric field directs the PPi-Dye toward the negativeelectrode 71B, while the intact γ-dNTP-Dye molecules move toward thepositive electrode 71A. Thereafter, the PPi-Dye molecules are detectedin the transverse channel. Advantageously, this embodiment reduces oreliminates background from intact γ-dNTP-Dye molecules, thus allowingthe use of high γ-dNTP-Dye concentrations to drive the polymerasereaction.

As such, the present invention provides a method for separating alabeled nucleotide phosphate having a detectable moiety from a releasedcharged detectable moiety in a sample stream, comprising: a)immobilizing a nucleic acid complex onto a solid support in a singlemolecule configuration; b) contacting the complex with a polymerase anda plurality of nucleotide phosphates, wherein at least one of theplurality of nucleotide phosphate has a detectable moiety, wherein thedetectable moiety is released as a charged detectable moiety when the NPis incorporated into the primer nucleic acid; and c) applying anelectric field to the sample stream, thereby separating the labeled NPfrom the charged detectable moiety.

In another embodiment, the present invention provides a method forsequencing a target nucleic acid comprising: a) immobilizing a nucleicacid polymerase onto a solid support in a single molecule configuration,wherein the solid support is disposed in a flowcell having an inlet portand an outlet port; b) contacting the solid support with a sample streamcomprising a target nucleic acid, a primer nucleic acid and a detectablenucleotide phosphate wherein the sample stream flows through theflowcell; c) applying an energy field to the sample stream; and d)detecting the detectable nucleotide phosphate thereby sequencing thetarget nucleic acid. Optionally, the primer nucleic acid is attached tothe target nucleic acid.

Suitable nucleobases include, but are not limited to, adenine, guanine,cytosine, uracil, thymine, deazaadenine and deazaguanosine. In apreferred embodiment, the NP probes are dNTP probes having charge switchcharacteristics. In other aspects, the nucleobase is immobilized on asolid support and the sample stream contains a polymerase.

In certain preferred embodiments, the intact NP probe has a firstmolecular charge associated therewith; and whereupon cleavage of theterminal phosphate as a terminal phosphate fluorophore moiety, thephosphate fluorophore moiety carries a second molecular charge, whereinthe difference between the first molecular charge and the secondmolecular charge is preferably between 1 and 4. The charge-switchcharacteristics are implemented upon enzymatic cleavage of the terminalphosphate or pyrophosphate group.

In certain aspects, at least two energy fields are used. By using atleast two energy fields, the signal/noise discrimination can be enhancedwhen designed in conjunction with the expected charge on the signalmolecule versus the noise molecule. That is, the signal molecule(fluorescent phosphates) responds more strongly to a particular field ifits charge magnitude exceeds that of the noise molecule (e.g.unincorporated fluorescently labeled nucleotides), or less strongly ifits charge magnitude is less than that of the noise molecule.

Upon incorporation by a polymerase, the dNTP is hydrolyzed as usual andthe liberated pyrophosphate-dye moiety diffuses into the surroundingmedium. The free dye molecule is fluorescent and its appearance isimaged at video-rate under a microscope. A flowing stream sweeps the dyeaway from the parent DNA molecule. As the polymerase continues to movealong the DNA, the nucleotide sequence is read from the order ofreleased dyes. Sequencing proceeds quickly, as fast as the polymeraseprogresses along the DNA template.

In another embodiment, the present invention provides a method forseparating an intact NP probe from a phosphate detectable moiety,comprising: a) providing a sample comprising an intact NP probe with adetectable moiety attached thereto, whereupon an enzymatic cleavage ofthe intact NP probe, which produces a phosphate detectable moiety, thephosphate detectable moiety carries a molecular charge which isdifferent than the molecular charge of the intact NP probe; and b)applying an energy field to the sample, thereby separating the phosphatedetectable moiety from the intact NP probe.

In still yet another embodiment, the present invention provides a methodfor sequencing a nucleic acid, comprising: providing a target nucleicacid, a primer strand, a polymerase, and a plurality of NP probes;mixing the target nucleic acid, the primer strand, the polymerase, theplurality of NP probes in a flowcell under conditions permitting targetdependent polymerization of the plurality of NP probes, therebyproviding a polymerization product; and separating the polymerizationproduct by an energy field in the flowcell to provide a sequence of thetarget nucleic acid.

In yet another embodiment, the present invention provides a method forsequencing a nucleic acid comprising: providing a target nucleic acid, apolymerase priming moiety, a polymerase, and labeled NPs; mixing thetarget nucleic acid, the polymerase priming moiety, the polymerase andthe labeled NPs under conditions permitting target dependentpolymerization of the NPs, such conditions which are capable ofproviding a time sequence of labeled pyrophosphate products; separatingby charge the phosphate detectable moieties products from unpolymerizedlabeled NPs; and, detecting over time the phosphate detectable moietiesto provide a sequence of the target nucleic acid. In certain aspects,the method relates to multi-molecule DNA sequencing, as well as singlecolor (multi-molecule or single-molecule) sequencing where fourdifferent NP's (all labeled with the same color) are sequentiallyintroduced to the reaction site. In other aspects, two, three, orfour-color sequencing can be used.

B. Detection of Pyrophosphate

In certain other embodiments, the present invention provides aheterogeneous assay for the detection of pyrophosphate. The detection ofpyrophosphate is advantageous in a number of biological reactions. Forexample, in a DNA polymerase reaction, wherein the polymerase selects asingle DNA molecule from solution and thereafter incorporates thenucleotide at the 3′-end of a primer strand, the natural consequence ofsuch incorporation is the release of pyrophosphate. If the assaysolution comprises the four deoxynucleotide triphosphates, each dNTPlabeled with a different color of fluorescent dye attached to theγ-phosphate, it is then possible to sequentially record the activity ofthe polymerase operating on a target DNA. The nucleotide sequence of thetarget DNA can thereafter be read directly from the order of releaseddyes attached to the pyrophosphate.

In other embodiments, the present invention provides methods fordetecting and identifying individual fluorogenic NP molecules such asdNTP molecules, as a polymerase incorporates them into a single nucleicacid molecule. In certain aspects, a fluorescent dye is attached to theγ-phosphate. As describe above, charged moieties are attached to thenucleobase to modulate a change in the electric charge associated withthe dye upon hydrolysis by a polymerase.

As such, the present invention provides a method for detectingpyrophosphate cleavage, the components of the assay comprising acharge-switch NTP, a target nucleic acid, a primer nucleic acid and apolymerase, the method comprising: (a) flowing the labeled charge-switchnucleotide phosphate (NP) having a γ-phosphate with a fluorophore moietyattached thereto, past an immobilized component selected from the groupconsisting of the polymerase and the target nucleic acid; (b)incorporating the NP on a primer strand hybridized to the target nucleicacid using an enzyme and releasing the γ-phosphate with the fluorophoremoiety attached thereto; and (c) detecting the fluorescent moietythereby detecting pyrophosphate cleavage. In the methods of the presentinvention, either the polymerase or the target nucleic acid is attachedto a solid phase, such as a solid support. Preferably, in the methods ofthe present invention, the nucleic acid is immobilized on a solidsupport.

In many of the embodiments herein, the methods of the present inventionemploy a DNA polymerase such as DNA polymerase I, II or III. In otheraspects, suitable polymerases include, but are not limited to, a DNAdependent RNA polymerase and reverse transcriptase such as an HIVreverse transcriptase. Specific examples include, but are not limitedto, T7 DNA polymerase, +29 DNA polymerase, T5 DNA polymerase, E. ColiDNA polymerase I, T4 DNA polymerase, T7 RNA polymerase and Taq DNApolymerase. Those of skill in the art will know of other enzymes orpolymerases suitable for use in the present invention. In certainaspects, the target nucleic acid is bathed in a flowing solutioncomprising: polymerase unlabeled, single-stranded DNA fragmentshybridized to an oligonucleotide primer and a mixture of NTPs.Optionally, the primer can be attached to the immobilized target nucleicacid.

In certain aspects, detection of the phosphate detectable moiety (e.g.,PPi-Dye) is accomplished using an enzyme coupled assay. PPi can bedetermined by many different methods and a number of enzymatic methodshave been described in the literature (Reeves et al., (1969), Anal.Biochem., 28, 282-287; Guillory et al., (1971), Anal. Biochem., 39,170-180; Johnson et al., (1968), Anal. Biochem., 15, 273; Cook et al,(1978), Anal. Biochem. 91, 557-565; and Drake et al., (1979), Anal.Biochem. 94, 117-120). Those of skill in the art will know of otherenzyme coupled assays suitable for use in the present invention.

In one embodiment, the use of a phosphatase enhances the charge-switchmagnitude by dephosphorylating the PPi-F. In certain other aspects, itis preferred to use luciferase and luciferin in combination to identifythe release of pyrophosphate since the amount of light generated issubstantially proportional to the amount of pyrophosphate releasedwhich, in turn, is directly proportional to the amount of baseincorporated. The amount of light can readily be estimated by a suitablelight sensitive device such as a luminometer.

Luciferin-luciferase reactions to detect the release of PPi are wellknown in the art. In particular, a method for continuous monitoring ofPPi release based on the enzymes ATP sulphurylase and luciferase hasbeen developed by Nyren and Lundin (Anal. Biochem., 151, 504-509, 1985)and termed ELIDA (Enzymatic Luminometric Inorganic PyrophosphateDetection Assay). The foregoing method may be modified, for example, bythe use of a more thermostable luciferase (Kaliyama et al., 1994,Biosci. Biotech. Biochem., 58, 1170-1171). The preferred detectionenzymes involved in the PPi detection reaction are thus ATP sulphurylaseand luciferase.

As shown in FIG. 8, in preferred compounds of the present invention,wherein a fluorophore is attached to the γ-phosphate, the fluorophore isreleased from the nucleotide along with the pyrophosphate group. Usingsingle molecule detection for example, fluorescent signals appear at thelocations of the individual molecules being observed. In certainaspects, each type of nucleotide is labeled with a different fluorophoreso that the incorporated nucleobases can be sequentially identified bythe released fluorophores. Preferably, the nucleotide triphosphate (NTP)of the present methods include, but are not limited to, deoxyadenosinetriphosphate, deoxycytosine triphosphate, deoxyguanosine triphosphate,deoxythymidine triphosphate, deoxyuridine triphosphate or mixturesthereof, each with a unique fluorophore attached to the γ-phosphate.

In certain embodiments, an unlabeled, single-stranded target nucleicacid with a primer hybridized thereto is tethered to the surface of asolid support such as a glass slide. In another aspect, a doublestranded nucleic acid with a nick is tethered. An-aqueous solutioncomprising an enzyme, such as a DNA polymerase, and fluorogenic dNTPsflows across the surface. Alternatively, in another embodiment, anindividual polymerase molecule is immobilized on a glass slide and thepolymerase is bathed in a flowing solution comprising: 1) unlabeled,single-stranded DNA fragments hybridized to an oligonucleotide primer(or a covalently attached hairpin) and 2) a mixture of deoxynucleotidetriphosphates, each uniquely labeled with a different color offluorescent dye attached to the 7-phosphate.

In certain embodiments, an evanescent light field is set up by totalinternal refection (TIR) of a laser beam at the glass-aqueous solutioninterface. In certain aspects, the TIR illumination field iscontinuously imaged at video-rate with an intensified charge coupledevice (ICCD) camera.

C. Solid Phase

In certain embodiments herein, the present invention relates to methodswherein a material in the solid-phase interacts with reagents in theliquid phase. In certain aspects, the nucleic acid is attached to thesolid phase. The nucleic acid can be in the solid phase such asimmobilized on a solid support, through any one of a variety ofwell-known covalent linkages or non-covalent interactions. In certainaspects, the support is comprised of insoluble materials, such ascontrolled pore glass, a glass plate or slide, polystyrene, acrylamidegel and activated dextran. In other aspects, the support has a rigid orsemi-rigid character, and can be any shape, e.g. spherical, as in beads,rectangular, irregular particles, gels, microspheres, or substantiallyflat support. In some embodiments, it can be desirable to create anarray of physically separate sequencing regions on the support with, forexample, wells, raised regions, dimples, pins, trenches, rods, pins,inner or outer walls of cylinders, and the like. Other suitable supportmaterials include, but are not limited to, agarose, polyacrylamide,polystyrene, polyacrylate, hydroxethylmethacrylate, polyamide,polyethylene, polyethyleneoxy, or copolymers and grafts of such. Otherembodiments of solid-supports include small particles, non-poroussurfaces, addressable arrays, vectors, plasmids, orpolynucleotide-immobilizing media.

As used in the methods of the present invention, nucleic acid can beattached to the solid support by covalent bonds, or other affinityinteractions, to chemically reactive functionality on thesolid-supports. The nucleic acid can be attached to solid-supports attheir 3′, 5′, sugar, or nucleobase sites. In certain embodiments, the 3′site for attachment via a linker to the support is preferred due to themany options available for stable or selectively cleavable linkers.Immobilization is preferably accomplished by a covalent linkage betweenthe support and the nucleic acid. The linkage unit, or linker, isdesigned to be stable and facilitate accessibility of the immobilizednucleic acid to its sequence complement. Alternatively, non-covalentlinkages such as between biotin and avidin or streptavidin are useful.Examples of other functional group linkers include ester, amide,carbamate, urea, sulfonate, ether, and thioester. A 5′ or 3′biotinylated nucleotide can be immobilized on avidin or streptavidinbound to a support such as glass.

In other aspects of the methods of the present invention, the polymeraseis immobilized on a solid support. Suitable solid supports include, butare not limited to, controlled pore glass, a glass plate or slide,polystyrene, and activated dextran. In other aspects, synthetic organicpolymers such as polyacrylamide, polymethacrylate, and polystyrene arealso illustrative support surfaces. In addition, polysaccharides such ascellulose and dextran, are further illustrative examples of supportsurfaces. Other support surfaces such as fibers are also operable.

In other aspects, polymerase immobilization is accomplished using solidchromatography resins that have been modified or activated to includefunctional groups that permit the covalent coupling of resin to enzyme.Typically, aliphatic linker arms are employed. The enzymes of thepresent invention can also be noncovalently attached to a solid supportsurface, through, for example, ionic or hydrophobic mechanisms.

In a preferred embodiment, covalent attachment of a protein or nucleicacid to a glass or metal oxide surface can be accomplished by firstactivating the surface with an amino silane. DNA or protein derivatizedwith amine-reactive functional groups can then attach to the surface(see, K. Narasimhan et al., Enzyme Microb. Technol. 7, 283 (1985); M. J.Heller et al., U.S. Pat. No. 5,605,662; and A. N. Asanov et al., Anal.Chem. 70, 1156 (1998)).

The ordinarily skilled artisan will know numerous other schemes forlinking nucleic acid and proteins to support surfaces. Moreover, thechoice of support surface and the method of immobilizing the enzyme islargely a matter of convenience and depends on the practitioner'sfamiliarity with, and preference for, various supports surfaces, as wellas preference for various immobilizing schemes, and knowledge of thesubstrate.

In operation, when the enzyme is immobilized, such as a DNA polymerase,the enzyme selects a single DNA molecule from solution. The polymeraseincorporates a first nucleotide at the 3′-end of the primer strand. Thepolymerase then translocates to the next position on the target DNA,incorporates a complementary nucleotide, and releases the respectivePPi-Dye. The released dyes move away from the immobilized enzyme in theflowing sample solution. These events can then be recorded sequentiallyby video-rate imaging using for example, a CCD camera, capable ofdetecting single fluorophore molecules. The resulting movie shows theactivity of a single polymerase molecule operating on a single moleculeof DNA. The nucleotide sequence of the DNA target is read directly fromthe order of released dyes. When the first nucleic acid molecule hasbeen sequenced, the polymerase releases it and selects another templatefrom solution. Many DNA molecules are thereby sequenced by a singlepolymerase. The process continues for the life of the enzyme.

D. Preparation of Target Nucleic Acid The target nucleic acid can beprepared by various conventional methods. For example, target nucleicacid can be prepared as inserts of any of the conventional cloningvectors, including those used in conventional DNA sequencing. Extensiveguidance for selecting and using appropriate cloning vectors is found inSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Laboratory, New York, 1989), and like references.Sambrook et al. and Innis et al., editors, PCR Protocols (AcademicPress, New York, 1990) also provide guidance for using polymerase chainreactions to prepare target polynucleotides. Cloned or PCR-amplifiedtarget nucleic acid is prepared which permit attachment to solidsupports.

In a preferred embodiment, sheared DNA fragments from a subjectorganism, preferably human, are treated to provide blunt ends, thenligated to two oligodeoxynucleotides (ODNs). The first ODN isderivatized with biotin and the second is complementary to a sequencingprimer. The ligated DNA is denatured, it is brought into contact with astreptavidin-activated slide, and it attaches through the biotin to theslide. A primer is hybridized to the tethered fragments prior tosequencing. Only DNA fragments having each type of ODN can both attachand be sequenced; fragments having two primer ODNs will not attach, andthose having two attachment ODNs will not prime. DNA attachment couldalso be accomplished by direct covalent coupling as practiced on DNAchips (see, U.S. Pat. No. 5,605,662). Unlike DNA chips that require adense lawn of probes, preferably, a few DNA molecules are bound per unitsurface area. Binding density is easily controlled by adding a carrierto the DNA sample (e.g., free biotin to a biotinylated DNA sample).

The primers (DNA polymerase) or promoters (RNA polymerase) aresynthetically made using conventional nucleic acid synthesis technology.The complementary strands of the probes are conveniently synthesized onan automated DNA synthesizer, e.g. an Applied Biosystems, Inc. (FosterCity, Calif.) model 392 or 394 DNA/RNA Synthesizer, using standardchemistries, such as phosphoramidite chemistry, e.g. disclosed in thefollowing references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311(1992); Molko et al, U.S. Pat. No. 4,980,460; Koster et al, U.S. Pat.No. 4,725,677; Caruthers et al, U.S. Pat. Nos. 4,415,732; 4,458,066; and4,973,679; and the like. Alternative chemistries, e.g. resulting innon-natural backbone groups, such as phosphorothioate, phosphoramidate,and the like, may also be employed provided that the resultingoligonucleotides are compatible with the polymerase. They can be orderedcommercially from a variety of companies, which specialize in customoligonucleotides.

Purification of oligonucleotides, where necessary, is typicallyperformed by either native acrylamide gel electrophoresis or byanion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom.255:137-149. The sequence of the synthetic oligonucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology 65:499-560.

While primers can hybridize to any of a number of sequences, selectingoptimal primers is typically done using computer assisted considerationof available sequences and excluding potential primers which do not havedesired hybridization characteristics, and/or including potentialprimers which meet selected hybridization characteristics. This is doneby determining all possible nucleic acid primers, or a subset of allpossible primers with selected hybridization properties (e.g., thosewith a selected length, G:C ratio, uniqueness in the given sequence, andthe like.) based upon the known sequence. The selection of thehybridization properties of the primer is dependent on the desiredhybridization and discrimination properties of the primer.

One of skill is thoroughly familiar with the theory and practice ofnucleic acid hybridization and primer selection. Gait, ed.Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford(1984); W. H. A. Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K.L. Dueholm J. Org. Chem. 59, 5767-5773 (1994); S. Agrawal (ed.) Methodsin Molecular Biology, volume 20; and Tijssen (1993) LaboratoryTechniques in biochemistry and molecular biology-hybridization withnucleic acid probes, e.g., part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,N.Y. provide a basic guide to nucleic acid hybridization. Innis supraprovides an overview of primer selection.

Primers in combination with polymerases are used to sequence target DNA.Primer length is selected to provide for hybridization to complementarytemplate DNA The primers will generally be at least 10 bp in length,usually at least between 15 and 30 bp in length. Primers are designed tohybridize to known internal sites on the subject target DNA.Alternatively, the primers can bind to synthetic oligonucleotideadaptors joined to the ends of target DNA by a ligase. Similarly wherepromoters are used, they can be internal to the target DNA or ligated asadaptors to the ends.

The reaction mixture for the sequencing comprises an aqueous buffermedium, which is optimized for the particular polymerase. In general,the buffer includes a source of monovalent ions, a source of divalentcations and a buffering agent. Any convenient source of monovalent ions,such as KCl, K-acetate, NH₄-acetate, K-glutamate, NH₄Cl, ammoniumsulfate, and the like may be employed, where the amount of monovalention source present in the buffer will typically be present in an amountsufficient to provide for a conductivity in a range from about 500 to20,000, usually from about 1000 to 10,000, and more usually from about3,000 to 6,000 microhms.

The divalent cation may be magnesium, manganese, zinc and the like,where the cation will typically be magnesium. Any convenient source ofmagnesium cation may be employed, including MgCl₂, Mg-acetate, and thelike. The amount of Mg ion present in the buffer may range from 0.5 to20 mM, but will preferably range from about 1 to 12 mM, more preferablyfrom 2 to 10 mM and will ideally be about 5 mM.

Representative buffering agents or salts that may be present in thebuffer include Tris, Tricine, HEPES, MOPS and the like, where the amountof buffering agent will typically range from about 5 to 150 mM, usuallyfrom about 10 to 100 mM, and more usually from about 20 to 50 mM, wherein certain preferred embodiments the buffering agent will be present inan amount sufficient to provide a pH ranging from about 6.0 to 9.5,where most preferred is pH 7.6 at 25° C. Other agents which may bepresent in the buffer medium include chelating agents, such as EDTA,EGTA and the like.

E. Detection

In certain embodiments, the enzymatic reaction is monitored using singlemolecule detection. The single-molecule fluorescence detection of thepresent invention can be practiced using optical setups includingnear-field microscopy, far-field confocal microscopy, wide-fieldepi-illumination, and total internal reflection fluorescence (TIRF)microscopy. Suitable photon detectors include, but are not limited to,photodiodes and intensified CCD cameras. In other embodiments, videochips such as CMOS chips can be used. In a preferred embodiment, anintensified charge couple device (ICCD) camera is used. The use of aICCD camera to image individual fluorescent dye molecules in a fluidnear the surface of the glass slide is advantageous for several reasons.With an ICCD optical setup, it is possible to acquire a sequence ofimages in time (movies) of fluorophores. In certain aspects, each of theNTPs of the present invention has a unique fluorophore associated withit, as such, a four-color instrument can be used having four cameras andup to four excitation lasers or any combination thereof. Thus, it ispossible to use this optical setup to sequence DNA. In addition, manydifferent DNA molecules can be imaged and sequenced simultaneously.Moreover, with the use of image analysis algorithms, it is possible totrack the path of single dyes and distinguish them from fixed backgroundfluorescence.

In certain aspects, the preferred geometry for ICCD detection ofsingle-molecules is total internal reflectance fluorescence (TIRF)microscopy. In TIRF, a laser beam totally reflects at a glass-waterinterface. The field does not end abruptly at the reflective interface,but its intensity falls off exponentially with distance. The thin“evanescent” optical field at the interface provides low background andenables the detection of single molecules with signal-to-noise ratios ofabout 6:1, preferably about 8:1 and more preferably about 12:1 atvisible wavelengths (see, M. Tokunaga et al., Biochem. and Biophys. Res.Comm. 235, 47 (1997) and P. Ambrose, Cytometry, 36, 244 (1999)). Incertain aspects, the TIR illumination field is continuously imaged atvideo-rate with an intensified charge couple device (ICCD) camera. It isthus possible to image the pyrophosphate as it is hydrolyzed by theenzyme.

The penetration of the field beyond the glass depends on the wavelengthand the laser beam angle of incidence. Deeper penetrance is obtained forlonger wavelengths and for smaller angles to the surface normal withinthe limit of a critical angle. In typical assays, fluorophores aredetected within about 200 nm from the surface, which corresponds to thecontour length of about 600 base pairs of DNA. Either Prism-type orobjective type TIRF geometry is for single-molecule imagining is used.(see, X-H. N. Xu et al., Science, 281, 1650 (1998) and Tokunaga et al.,Biochem Biophys. Research Comm., 235, 47 (1999)).

DNA, proteins and lipids have all been detected in complex samples withsingle-molecule sensitivity using labeled probes (see, L. Edman et al.,Proc. Natl. Acad. Sci. USA, 93, 6710 (1996); M. Kinjo et al., NucleicAcids Res. 23, 1795 (1995); A. Castro and J. G. K. Williams, Anal. Chem.69, 3915 (1997); S, Nie, et al., Science 266, 1018 (1994); S, Nie, etal., Anal. Chem. 67, 2849 (1995); and T. Schmidt et al., Proc. Natl.Acad. Sci. USA 9, 2926 (1996)). In addition to simple detection, singlefluorophores are also characterized with respect to fluorescencelifetime, spectral shifts and rotational orientation. In a preferredaspect of the present invention, an aqueous solution comprising anenzyme, such as a DNA polymerase, and distinguishable fluorogenic dNTPs,i.e., a characteristic dye for each nucleobase, flows across thesurface. An evanescent light field is set up by total internal refection(TIR) of a laser beam at the glass-aqueous solution interface. Incertain aspects, the TIR illumination field is continuously imaged atvideo-rate with an intensified charge couple device (ICCD) camera. It isthus possible to image the pyrophosphate as it is hydrolyzed by theenzyme.

Upon incorporation by polymerase, the dNTP is hydrolyzed as usual andthe liberated terminal phosphate (e.g., pyrophosphate-dye) moietydiffuses into the surrounding medium. The free dye molecule, is imagedat video-rate under a microscope. A flowing stream sweeps the dye awayfrom the parent DNA molecule. As the polymerase continues to move alongthe DNA, the nucleotide sequence is read from the order of releaseddyes. Sequencing proceeds quickly, as fast as the polymerase progressesalong the DNA template.

In another embodiment, the present invention includes sensors asdisclosed in U.S. Pat. No. 5,814,524, which issued to Walt et al., onSep. 29, 1998. An optical detection and identification system isdisclosed therein that includes an optic sensor, an optic sensingapparatus and methodology for detecting and evaluating one or moreanalytes or ligands of interest, either alone or in mixtures. The systemis comprised of a supporting member and an array formed ofheterogeneous, semi-selective polymer films which function as sensingreceptor units and are able to detect a variety of different analytesand ligands using spectral recognition patterns. Using this system, itis possible to combine viewing and hemical sensing with imaging fiberchemical sensors.

In yet another embodiment, the detection is accomplished using blockadecurrent, as described in U.S. Pat. No. 5,795,782 issued to Church etal., and which is incorporated herein by reference in its entirety forall purposes. As disclosed therein, two pools of medium used may be anyfluid that permits adequate analyte mobility for interface interaction.Typically, the pools will be liquids, usually aqueous solutions or otherliquids or solutions in which the analyte can be distributed. Theinterface between the pools is designed to interact sequentially withthe analyte molecule one at a time. The useful portion of the interfacemay be a passage in or through an otherwise impermeable barrier, or itmay be an interface between immiscible liquids. It is preferable thatonly one passage is present or functional in the impermeable barrier.The interface-dependent measurements can be any measurement, e.g.,physical or electrical, that varies with analyte-interface interaction.For example, physical changes the analyte cause as they interactsequentially with the interface may be measured. Current changesresulting from the analyte's interference with ion flow at the interfacemay be measured. The measurements may reflect the sequential interactionof the analyte with the interface, so as to permit evaluation ofsequence-dependent characteristics.

In one embodiment, the pools include electrically conductive medium,which can be of the same or different compositions. The pools withconducting media are separated by an impermeable barrier containing anion-permeable passage, and measurements of the interface characteristicsinclude establishing an electrical potential between the two pools suchthat ionic current can flow across the ion permeable passage. When theanalyte interacts sequentially with the interface at the ion permeablepassage, the ionic conductance of the passage will change (e.g.,decrease or increase) as each analyte interacts.

The conducting medium used can be any medium, preferably a solution,more preferably an aqueous solution, which is able to carry electricalcurrent. Such solutions generally contain ions as the current conductingagents, e.g., sodium, potassium, chloride, calcium, cesium, barium,sulfate, and phosphate. Conductance (g) across the pore or channel isdetermined by measuring the flow of current across the pore or channelvia the conducting medium. A voltage difference can be imposed acrossthe barrier between the pools by conventional means, e.g., via a voltagesource, which injects or administers current to at least one of thepools to establish a potential difference. Alternatively, anelectrochemical gradient may be established by a difference in the ioniccomposition of the two pools, either with different ions in each pool,or different concentrations of at least one of the ions in the solutionsor media of the pools. In this embodiment of the invention, conductancechanges are measured and are indicative of analyte-dependentcharacteristics.

F. High Throughput Screening The present invention also providesintegrated systems for high-throughput screening of DNA sequencing andpyrophosphate detection. The systems typically include robotic armature,which transfers fluid from a source to a destination, a controller thatcontrols the robotic armature, an ICCD camera, a data storage unit whichrecords the detection, and an assay component such as a microtiter dishor a substrate comprising a fixed reactant. A number of robotic fluidtransfer systems are available, or can easily be made from existingcomponents. For example, a Zymate XP (Zymark Corporation; Hopkinton,Mass.) automated robot using a Microlab 2200 (Hamilton; Reno, Nev.)pipetting station can be used to transfer parallel samples to set upseveral parallel simultaneous polymerase reactions.

Optical images viewed (and, optionally, recorded) by a camera or otherrecording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e.g., bydigitizing the image and storing and analyzing the image on a computer.A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical image. In certain aspects, the integrated system ofthe present invention carries light from the specimen field to thecharge-coupled device (CCD) camera, which includes an array of pictureelements (pixels). The light from the specimen is imaged on the CCDcamera. Particular pixels corresponding to regions of the specimen(e.g., individual polymerase sites on a glass surface) are sampled toobtain light intensity readings for each position. Multiple pixels areprocessed in parallel to increase speed. The apparatus and methods ofthe invention are easily used for viewing any sample, e.g., byfluorescent or dark field microscopic techniques.

IV. Systems

FIG. 9 is a schematic of a microfabricated flowcell system 90 of thepresent invention. This diagram is merely an illustration and should notlimit the scope of the claims herein. One of ordinary skill in the artwill recognize other variations, modifications, and alternatives.

As shown therein, the present invention provides a microfabricatedflowcell 90 having an inlet port 91 and an outlet port 92 wherein asample stream having a detectable analyte flows therethrough. In certainaspects, the system includes at least a first energy field source 93having an energy field transverse to the sample stream. In someembodiments, the system comprises a second energy field source 94 havinga second energy field axial to the sample stream. The transverse fieldhas a pair of electrodes 95 a, 95 b and optionally, the axial field hasa hydrostatic pressure differential 96 a, 96 b. The system also includesa detector 99 for detecting the analyte in a microchannel zone 98.Suitable energy fields include, but are not limited to, an electricfield, a thermal field, a magnetic field, an electromagnetic field, aphotoelectric field, a light field, a mechanical field, a pressure fieldor combinations thereof. Preferably electric and pressure fields areemployed.

In certain embodiments, the flowcell is fabricated by microfabricationmethods known to those of skill in the art. For example, precisioninjection molded plastics or molded elastomers can also be used forfabrication. The flowchamber can be made of plastic or glass and shouldeither be open or transparent in the plane viewed by the detector,microscope or optical reader.

In one embodiment, the flowcell is about 0.1 mm to about 100 cm inlength, preferably about 1 mm to about 10 cm in length. In certainaspects, the flowcell has channels for the sample stream that can be ofdifferent dimensions and are typically about 0.5-10 cm in length andhave a depth of 0.5-100 μm. Channel dimensions can vary from place toplace within the same flowcell. The shape of the channel can vary andcan be rectangular, oval, circular, triangular, trapezoidal orotherwise. In certain aspects, various channel shapes are present. Thewidth of the channel is typically about 1 μm to about 100 μm.

In certain embodiments, the system may also include an analyte streamintroduced into the inlet port 91 comprising a liquid carrier containingsubstrate particles, nucleotides, enzymes, and the like. In certainembodiments, the analyte is immobilized on a solid support such as abead, and the bead may be trapped on a feature in the microchannel. Theliquid carrier can be any fluid capable of accepting particles from afeed stream and containing an indicator substance. Preferred samplestreams comprise water and solutions such as salt water with bufferedsolution well known to those of skill in the art. Alternatively, variousorganic solvents are suitable such as acetone, isopropyl alcohol,ethanol, or any other liquid convenient that does not interfere withdetection.

As disclosed in PCT publication No. WO 00/36152 and incorporated hereinby reference, in a preferred embodiment, each nucleotide has a uniquefluorophore associated with it, as such, a four-color instrument can beused having four cameras and four excitation lasers, or one camera withan image splitter device, or less than four excitation lasers assufficient to excite the four different dyes. Thus, it is possible touse this optical setup to sequence DNA. In addition, many different DNAmolecules immobilized in microchannels can be imaged and sequencedsimultaneously. Moreover, with the use of image analysis algorithms, itis possible to track the path of single dyes and distinguish them fromfixed background fluorescence.

In an alternative embodiment, the nucleotides disclosed in U.S. Pat. No.6,232,075, issued Mar. 15, 2001 to Williams, and which is incorporatedherein by reference in its entirety for all purposes, can be used. Asdisclosed therein, nucleotide probes having fluorescent labeled attachedthereto are disclosed.

In certain other embodiments, detection and analysis is done by variousmethods known to the art, including optical means, such as opticalspectroscopy, and other means such as absorption spectroscopy, Ramanspectroscopy or fluorescence, by chemical indicators which change coloror other properties when exposed to the analyte, by immunological means,electrical means, e.g., electrodes inserted into the device,electrochemical means, blockade current means, radioactive means, orvirtually any microanalytical technique known to the art to detect thepresence of an analyte such as an ion, molecule, polymer, virus, nucleicacid sequence, antigen, microorganism, and the like. Preferably opticalor fluorescent means are used, and antibodies, nucleotides and the likeare attached to fluorescent markers.

In certain other embodiments, the flowcell system of the presentinvention further optionally comprises voltage probes, conductivityelectrodes, pH cells, conductivity meters, pH meters, ammeters,voltmeters, flowrate monitors, a data acquisition system and amicrocomputer. Those of skill in the art will recognize usefuladditional sensors and probes.

FIG. 10 is a schematic of a microfabricated flowcell system of thepresent invention having a plurality of flowcells (an array offlowcells). This diagram is merely an illustration and should not limitthe scope of the claims herein. One of ordinary skill in the art willrecognize other variations, modifications, and alternatives.

In this embodiment, the flowcell system 1000 of the present invention isextended into an array of flowcells 1072 a, 1072 b with a plurality ofcomponents. As used herein array is at least two flowcells. Forinstance, it is possible to have multiple immobilization sites, inletand outlet ports, detectors and the like.

V. Examples

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example I

This example shows an algorithm for calculating charge on the intactnucleotide probe and cleavage product, taking into account contributionsfrom hydrogen ions and divalent metal cations. It will be appreciated bythose of skill in the art that particular equilibrium constants areaffected by the instant chemical environment, and that the equilibriumconstants affect the outcome of the calculation. The algorithm explainedin the example was implemented in a computer program to execute thecalculations, some results of which are illustrated in FIG. 3.

For steps 1-3 below, refer to FIG. 5 for the definition of the indicatedequilibrium constants K (in boxes). Values for the equilibrium constantsare taken from analogous compounds in Frey and Stuhr (1972) Journal ofthe American Chemical Society, 94:8898. In the calculation steps, thesedefinitions apply: N-PPP-F “L” (ligand); H⁺=“H” (hydrogen ion) andMg⁺⁺=“M” (counter ion metal).

I. Step 1 K_(HL) Compute H Binding to L at a Given pH: The Equilibrium

L+H

LH

K _(HL) =[LH]/[L]*[H]

VALUE OF K_(HL)

LOG K_(HL)

2the primary ionization of all nucleotide mono, di and triphosphates

f1, the Fraction of L in Protonated Form LH

f1=[H]([H]+K _(HL))

f1

0

f2, the Fraction of L in Unprotonated Form L

f2=1−f1

f2

1 at neutral pH

II. Step 2-K_(ML) Compute M Binding to L at a Given [M] at the of Step 1The Equilibrium

L+M

LM

K _(ML) =[LM]/[L]*[M]

Value of K_(ML)

log K_(ML)=2.18

the analog of N-PPP-F is protonated-NTP (NTP-H), where H≅F

ATP-H+Mg

ATP(Mg)-H, log K=2.18

CTP-H+Mg

CTP(Mg)-H, log K=2.18

f5, the Fraction of L in Complexed Form ML

f5=[M]/([M]+K _(ML))

f6, the Fraction of L in Uncomplexed Form L

f6=1−f5

III. Step 3 K_(MHL)

Compute m binding to hl at a given [M] at the pH of step 1

The Equilibrium

HL+M

MHL

K _(MHL) =[MHL]/[HL]*[M]

Value of KMHL

[HL] is negligible[HL] is negligible because at neutral pH values, L is in theunprotonated form (K_(HL)≅2; above)

IV. Step 4 Compute the Fraction of Each Complexed Form of L (Refer toFIG. 5)

fracL=f2*f6

fracML=f2*f5

fracHL=f1*f4

0*f4

0

fracMHL=f1*f3

0*f3

0

V. Step 5 Compute Phosphate Charge Q_(phos) Averaged Over all Forms of L

Q _(phos) =Q(L)*fracL+Q(ML)*fracML+Q(HL)*fracHL+Q(MHL)*fracMHL

where each Q is defined in the FIG. 5

VI. Step 6

Compute Nucleobase Charge Q_(B) Due to Nucleobase Adducts B

The Equilibrium

B+H

BH

KBH=[BH]/[B]*[H]

value of K_(BH)depends on whether the adduct B is for example a carboxylate(−) orarginine(+) or quaternary amine(+)

log K_(BH)=4.5 for carboxylate

log K_(BH)=12 for arginine or quaternary ammonium salt

note: at pH values 6.5-8.5, these groups are effectively fixed negativeor positive charges

Fraction of Protonated and Unprotonated Forms

fracBH=[H]/([H]+K _(BH))

fracB=1−fracBH

Charge

Q_(B)=charge of B

Q_(BH)=charge of BH

Q _(B)=fracBH*Q _(BH)+fracB*Q _(B)

VII. Step 7

Compute γ-Label Charge Q_(G) from γ-Label Adducts Gsame logic as step 6

Q _(G)=fracGH*Q _(GH)+fracG*Q _(G)

VIII. Step 8 Compute Overall Charge Q_(N-PPP-F) on N-PPP-F

Q _(N-PPP-F) =Q _(phos) +Q _(B) +Q _(G)

Similar to the above, equilibrium calculations can be done for PP-F. Thesame logic applies as for N-PPP-F, except that different equilibriumconstants are used which are appropriate for PP-F (values given in boxesbelow). For steps 1-3 below, refer to FIG. 5 for the definition of theindicated equilibrium constants K (in boxes). Values for the equilibriumconstants are taken from analogous compounds in Frey and Stuhr (1972)JACS 94:8898. The following definitions apply PP-F=“L” (ligand); H⁺=“H”(hydrogen ion) and Mg⁺⁺=“M” counter ion or (metal).

I. Step 1-K_(HL) Compute H Binding to L at a Given pH: The Equilibrium

L+H

LH

K _(HL) =[LH]/[L]*[H]

-   -   VALUE OF K_(HL)        log K_(HL)        2        the primary ionization of all nucleotide mono, di and        triphosphates

f1, the Fraction of L in Protonated Form LH

f1=[H]([H]+K _(HL))

f1

0 at neutral pH

f2, the Fraction of L in Unprotonated Form L

f2=1−f1

f2

1 at neutral pH

II. Step 2-K_(ML)

Compute M Binding to L at a Given [M] at the pH of Step 1

The Equilibrium

L+M

LM

K _(ML) =[LM]/[L]*[M]

Value of K_(ML)

log K_(ML)=3.20

the analog of PP-F is unprotonated nucleotide diphosphate or protonatedpyrophosphate

ADP+Mg

ADP(Mg), log K=3.22

DP+Mg

CDP(Mg), log K=3.21

PP-H+Mg

(Mg)PP-H, log K=3.18

f5, the Fraction of L in Complexed Form ML

f5=[M]/([M]+K_(ML))

f6, the Fraction of L in Uncomplexed Form L

f6=1−f5

III. Step 3-K_(MHL) Compute M Biding to HL at a Given [M] at the pH ofStep 1 The Equilibrium

HL+M

K_(MHL)

K _(MHL) =[MHL]/[HL]*[M]

Value of K_(MHL)

log K_(MHL)=1.60

the analog of H-PP-F is protonated nucleotide diphosphate N-PP-H

ADP(H)+Mg

ADP(H)-Mg, log K=1.55

CDP(H)+Mg

ADP(H)-Mg, log K=1.60

f3, The Fraction of HL in Complexed Form MHL

f3=[M]/([M]+K _(MHL))

f4, The Fraction of HL in Uncomplexed Form HL

f4=1−f3

IV. Step 4 Compute the Fraction of Each Complexed Form of L (Refer toFigure)

fRacL=f2*f6

fracML=f2*f5

fracHL=f1*f4

fracMHL=f1*f3

V. Step 5 Compute Phosphate Charge Q_(phos) Averaged Over all Forms of L

Q _(phos) =Q(L)*fracL+Q(ML)*fracML+Q(HL)*fracHL+Q(MHL)*fracMHL

where each Q is defined in the Figure

VI. Step 6

Compute Nucleobase Charge Q_(B) Due to Nucleobase Adducts B

The Equilibrium

B+H

BH

KBH=[BH]/[B]*[H]

value of K_(BH)log K_(BH)=4.5 for carboxylatelog K_(BH)=12 for arginine or quaternary ammonium saltnote: at pH values 6.5-8.5, these groups are effectively fixed negativeor positive charges

Fraction of Protonated and Unprotonated Forms

fracBH=[H]/([H]+K _(BH))

fracB=1−fracBH

Charge

Q_(B)=charge of B

Q_(BH)=charge of BH

Q _(B)=fracBH*Q _(BH)+fracB*Q _(B)

VII. Step 7

Compute γ-Label Charge Q_(G) from γ-Label Adducts Gsame logic as step 6

Q _(G)=fracGH*Q _(GH)+fracG*Q _(G)

VIII. Step 8 Compute Overall Charge Q_(N-PPP-F) on N-PPP-F

Q _(N-PPP-F) =Q _(phos) +Q _(B) +Q _(G)

Example II Materials and Methods

Modeling was performed of nucleotide sequencing using the system of thepresent invention. The simulations were performed with MATLAB (TheMathWorks, Inc., Natick, Mass.) version R11.1, running on an IntelPentium III-based machine. The operating system is Windows 98 (secondedition). Nucleotide motion was calculated according to the followingmethod. For each time step,

-   -   1. Determine the voltage at this time for both pairs of plates        from a given waveform.    -   2. Calculate the electric field in the axial and transverse        directions due to the voltage. Add in a vector-wise fashion for        the total field.    -   3. Calculate the resultant velocity given the physical        parameters of the molecule. Note that the charge and diffusion        coefficient will be different for the quencher-nucleobase-dye        moiety than for the released pyrophosphate-dye moiety.    -   4. Given the time step, calculate the resultant motion from the        velocity.    -   5. Calculate a Gaussian-distributed movement due to diffusion.    -   6. Add the movement due to diffusion and the movement due to the        electric field in a vector-wise fashion.    -   7. Move the molecule.

A. This example is similar to an electrophoretic case, wherein a DCfield is applied axially.

In this example, no field is applied to the transverse plates, and aconstant field is applied to the axial plates. The charge on thedye-nucleobase-quencher structure is −4, and the charge on thepyrophosphate-dye structure is −2. The axial field strength is 3×10⁵V/m, which would result from, for example, 3000 V across 1 cm.

In this example, the charge ratio between the unincorporated structuresand the released dyes is 2, meaning that on average, the unincorporatedstructures travel twice as fast as the released dyes.

B. This example illustrates a DC field applied axially and an AC fieldapplied transversely.

In this example, the transverse field is modulated in a sinusoidalfashion. The charge ratio is 2. The axial field strength is the same asin Example 1, and the transverse field has a peak-to-peak amplitude of50 V. The frequency of oscillation is 200 Hz. The background moleculesspread or “throw” their photons over a larger area, since their spatialmodulation (peak-to-peak length of their paths) is greater due tostronger response to the E field. This has the effect of smoothing outthe background, resulting in a higher signal-to-noise ratio (SNR). Thepreferred setup is to have the released phosphate have a small, butdistinguishable path amplitude (if the amplitude were too great, itwould also scatter its photons over too many CCD pixels).

Example III

This example illustrates an AC field applied axially and an AC fieldapplied transversely.

Another case of interest is to have only AC components to the transverseand axial fields. If both field strengths are sinusoids comparable inamplitude and frequency, the resultant path coupled with diffusionpaints a bright spot on the image when an incorporation event occurs.This method allows a continual “wash” over the enzymes to encourageincorporation, while limiting the total traversal breadth to keepphotons concentrated in one place. Moreover, it is advantageous to usethis method when the CCD camera is reading out and the shutter isclosed, to avoid having signals travel far away during the “blackout”time (images are not being recorded). The peak axial field strength isthe same as the previous two DC examples, while the peak transversevoltage is 50 V. Both waveforms are at 200 Hz.

Example IV

This example illustrates an AC and DC field applied axially and an ACapplied transversely.

Applying both AC and DC fields to the axial plates results in shorter,fatter streaks since the molecules follow a spiral pattern down theflowcell. The DC component ensures eventual washing away of allmolecules. The AC field in the transverse direction encouragesunincorporated molecules to throw their photons over a larger area,which as before tends to smooth out the background.

Example V

This example illustrates that Mg⁺⁺ can change the electrophoreticmobility of dTTP and dTDP (unlabeled) from more negative to lessnegative. It also shows that the electrophoretic mobility ofdTTP-(++)-BODIPYTR can be changed from negative to positive as the Mg⁺⁺concentration increases.

5.1 Analysis of dTTP and dTDP by Capillary Electrophoresis

The effect of Mg⁺⁺ on the electrophoretic mobility of dTTP and dTDP wasdetermined by capillary electrophoresis. Electrophoresis buffercontained 50 mM Tris-acetate pH 8.0, 60 mM KCl, and variousconcentrations of MgCl₂ (3, 4, 6, 10, 15, 25 and 40 mM). The samplecontained 0.5 mM nucleotide (dTTP or dTDP; Sigma) and 0.8 mM mesityloxide (electroneutral marker; Sigma). The samples were analyzed bycapillary electrophoresis (Hewlett Packard) using an uncoated fusedsilica capillary (40 cm from injection end to detection zone). Voltagewas 8.5 kV and peaks were monitored by optical absorbance at 260 nm.Electrokinetic velocity of each sample peak was calculated by dividingdistance (40 cm) by elution time. Electroosmotic flow (EOF) of the bulkbuffer is taken as the velocity of the mesityl oxide marker. Nucleotideelectrophoretic velocity is the nucleotide electrokinetic velocity minusEOF. Nucleotide electrophoretic velocities are plotted as a function ofMg⁺⁺ concentration (FIG. 11A). The dTTP has a more negativeelectrophoretic mobility than the dTDP, as expected, because dTTP has anadditional phosphate group (“negative mobility” means that the moleculemoves like a negatively-charged molecule, towards the positiveelectrode). Mg⁺⁺ changed the electrophoretic mobility of bothnucleotides from more negative to less negative.

5.2 Analysis of dTTP-BOS(++)-BODIPYTR by Gel Electrophoresis

The net charge on dTTP was adjusted in a positive direction by addingtwo quaternary amine groups on a linker attached to the γ-phosphate; anelectroneutral dye marker was also attached to the linker, giving thecompound dTTP-BQS(++)-BODIPYTR (FIG. 4). The effect of Mg⁺⁺ on theelectrophoretic mobility of this nucleotide was determined by agarosegel electrophoresis. Electrophoresis buffer contained 50 mM Tris-acetatepH 8.0, 60 mM KCl, and various concentrations of MgCl₂ (0, 1, 1.5, 2, 3,4, 6, 10, 15, 25 and 40 mM). Slab gels (4% agarose) were cast in eachelectrophoresis buffer and each gel was placed in a slab gel apparatus(Bio-Rad) containing the respective electrophoresis buffer. The samplewells were loaded with 5 μL of 20 μM dTTP-BQS(++)-BODIPYTR andelectrophoresis was performed at 6 V per cm for 10 min. The gels werephotographed on a UV transilluminator and the separate images wereassembled into a single FIGURE (FIG. 11B). As was seen with unlabeleddTTP (FIG. 11A), Mg⁺⁺ added positive charge to the nucleotide. However,because the two quaternary amines make the labeled nucleotide lessnegative as compared to the unlabeled nucleotide, the effect of Mg⁺⁺ isto change the nucleotide mobility from net negative to net positive.

Example VI

This example illustrates the synthesis of a charge-switch nucleotide ofthe present invention.

6.1 Preparation of Compound 1′

In FIGS. 12A and B, 11.2 g of t-BOC anhydride (Aldrich, 218 g/mol, 52.4mmole) is dissolved in 100 mL of reagent grade methanol (Fisher). 10 mLof N,N dimethylpropyl amine (Aldrich, 102.1 g/mol, 48.9 mmole) is addedslowly to the reaction mixture. The reaction mixture is allowed to stirat room temperature for 16 hours. The reaction is deemed complete by TLC(C18, 1:1 Acetonitrile/Methanol, I₂ visualization) Solvent is evaporatedin vacuo. Compound 1′ (FIG. 15) is then purified bycolumn:chromatography (1:1 methylene chloride and methanol). Fractionscontaining pure compound 1 were combined and evaporated in vacuo toyield 17.7 g of purified product.

6.2 Preparation of Compound 2′

2.16 g of compound 1 (202 g/mol, 10.7 mmole) is dissolved in 10 mL dryreagent grade butyronitrile (Fisher). 1.51 grams of 1,4-diiodobutane isadded and the mixture is refluxed at 135° C. for 24 hours. The reactionis checked by TLC (C18, 40% aqueous methanol, I₂ visualization) anddetermined to be complete. The reaction mixture is precipitated withdiethyl ether and collected. After dissolving in methanol, the productis again precipitated with diethyl ether. The resultant viscous yellowresidue is dissolved in methanol and the solvent is removed in vacuo.This material is used without purification to form compound 3.

6.3 Preparation of Compound 3′

After drying, compound presents as fluffy yellow solid. This material isdissolved in 20 mL of SN HCl (prepared from concentrated HCl, Fisher)and stirred at room temperature for 5 hrs. Reaction completion ischecked by TLC (C18, 1:1 Acetonitrile/methanol, ninhydrin and UVvisualization). The acid solution is concentrated and product ofinterest is precipitated with diethyl ether. This solid is redissolvedin methanol and re-precipitated in diethyl ether. The solid is collectedand dried under vacuum. This product is used without furtherpurification or determination of yield.

6.4 Preparation of Compound 4′

1.1 mg of dTTP (Sigma, 492.7 g/mol, 2.2 μmole) is dissolved in 100 μL0.1 M MES pH 5.7. In a separate vial, 19.7 mg of EDC (Aldrich, 191.7g/mol, 100 μmole) is dissolved in 100 μL of 0.1 M MES pH 5.7. These twosolutions are combined and allowed to incubate at room temperature for10 minutes.

11.6 mg of compound 3 is dissolved in 400 μl of MES buffer. The pH ofthis solution is checked with pH strips and found to be 5.8. Thissolution is added to the activated nucleotide and the reaction isallowed to stand at room temperature for 110 minutes. The reaction ismonitored by reverse phase HPLC. The product of interest is isolated byreverse phase HPLC (C18, 4-80% Acetonitrile in 0.1 M TEAA over 20minutes). Solvent is removed from purified compound #4 in vacuo. Yieldis 12.7% from dTTP.

6.5 Preparation of Compound 56

Compound 4′ (0.15 μmole, 975 g/mol) is dissolved in 100 μl of 50 mMcarbonate buffer at pH 8.3. pH is checked by colorpHast pH strips andfound to be 8.3. 9.4 μL of 22 mM TAMRA-X-SE 6′ (Molecular Probes, 640.59g/mol, 0.23 μmole) is added and the reaction mixture is allowed to standat room temperature for 18 hours in the dark. Reaction is determinedcomplete after hydrolysis of all active dye ester. Product of interest56 is isolated by reverse phase HPLC (C18, 4-80% Acetonitrile in 0.1 MTEAA over 20 minutes). Solvent is removed from product in vacuo in thedark. Yield is 30% from compound 4.

Example VII

This example illustrates an assay system to demonstrate charge-switchingactivity of compounds of the present invention.

This assay is used to test for a change in the electric chargeassociated with a dye attached to the phosphate of a nucleotide. Thecharge switch on the dye is caused by cleavage of a phosphodiester bondthat links the dye to the nucleotide. In this example, cleavage iscatalyzed by snake venom phosphodiesterase.

Phosphodiesterase I (from Crotalus adamanteus venum; USB Corp.) wasdissolved in 110 mM Tris-HCl (pH 8.9) containing 110 mM NaCl, 15mMMgCl₂, to a final enzyme concentration of 40 units/mL. The reactionsample (50 μL) contained Phosphodiesterase I (3.6 units/mL), HEPES-NaOHbuffer (40 mM) and dTTP-BQS-BTR (38 μM). A control sample was the sameas the reaction sample except that the enzyme was omitted. The reactionand control samples were incubated at 37° C. for 1 hour and wereanalyzed by electrophoresis in a 5% agarose using a running buffercomprising 50 mM Tris-HCl pH 8.0, 60 mM KCl, 2 mM MgCl₂. The BTR dye inthe reaction sample migrated toward the negative electrode (−), whilethe dye in the control sample migrated toward the positive electrode(+).

Example VIII

This example demonstrates charge-switching activity of compounds of thepresent invention.

Materials

Microchannels were created by replica molding polydimethylsiloxane(PDMS) against a silicon master. The channels are 10 microns wide by 10microns deep. Two intersecting channels perpendicular to one anotherwere formed in the shape of a cross. The distal ends of each channelempty into separate circular wells of diameter 4 mm and depth 5 mm.

With reference to FIG. 9, platinum electrodes were placed into the wellsof the vertical channel 95 a, 95 b. (Although not explicitly shown inthe FIG. 9, the distal ends of each channel empty into separate circularwells i.e., 95 a, 95 b; and 96 a, 96 b. For this embodiment, thedesignations 95 a, 95 b and 96 a, 96 b are used interchangeably todenote the wells of the channels and the electrodes and pressure forcesrespectively). Voltage was applied across these electrodes to introducean electric field down the vertical channel. Simultaneously, pressurewas applied to both wells 96 a, 96 b. Pressure applied to well 96 aforces dye towards the cross intersection, while pressure applied towell 96 b prevents dye from continuing towards well 96 b (dye is forcedinto the vertical channel towards either well 95 a, 95 b.

The 530.9 line of a tunable argon-krypton laser was used to directlyilluminate the flowcell channels. A cleanup filter (center wavelength530 nm, bandwidth 10 nm) was inserted between the laser output and theflowcell. The emitted fluorescence was viewed with an invertedmicroscope using an air objective (10×, NA 0.25). After the objective aholographic notch filter (center wavelength 530.9 nm, bandwidth 3 nm)and a bandpass emission filter (center wavelength 575 nm, bandwidth 50nm) removed Rayleigh and Raman scatter. The resulting signal was imagedonto a CCD camera and captured onto a PC with frame grabber hardware andsoftware.

The buffer consisted of a mixture of 20 mM Tris-OAc pH 8, 3% (w/v)polyvinylpyrolidone (PVP), 2 mM MgCl₂, and 0.1% Tween 20. A buffer+dyesolution was formed by adding either gly-TAMRA (−1 charge) or BQS-TAMRA(+1 charge) dye to the same buffer constituents, such that the dyeconcentration was 1 μM.

The PDMS molds and borosilicate cover slips were treated in an oxygenplasma chamber for 1 minute. After treatment, upon contact the PDMS andglass would irreversibly bond. The plasma also causes the flowcell andglass surfaces to become hydrophilic, permitting easy filling of thechannels by capillary action.

I. The purpose of this experiment was to show that the negativelycharged dye could be forced to turn a corner by the application of anelectric field while suppressing electroosmotic flow (EOF).

Wells at the end of the vertical channels 95 a, 95 b, and at the end ofthe horizontal channel 96 b were filled with 40 μL of buffer solutionwhile watching the cross intersection on a monitor showing the magnifiedimage. After the channels were wetted, well 96 a was filled with 40 μLof buffer+dye solution. A pressure of 0.28 psi was applied to well 96 a,while simultaneously applying 0.43 psi to well 96 b. An electric fieldof 820 V/cm was applied from well 95 a to well 95 b (well 95 acontaining the positive electrode).

The electric field forces the negatively-charged dye towards well 95 a(the positive electrode). EOF is known to be suppressed since the dyedoes not move away from the positive electrode (EOF arising from anegatively-charged wall causes a bulk flow away from the positiveelectrode).

The polarity was then switched; an electric field equal in magnitude butopposite in direction was applied across wells at the end of thevertical channels. The dye switched direction so that it was againmoving towards the positive electrode.

II The purpose of this experiment was to show that positively charge dyealso could be forced to turn a corner by the application of an electricfield.

Wells 95 b, 95 a, and 96 b were filled with 40 μL of buffer solutionwhile watching the cross intersection on a monitor. After the channelswere wetted, well 96 a was filled with 40 μL of buffer+dye solution. Apressure of 0.88 psi was applied to well 96 a, while simultaneouslyapplying 1.09 psi to well 96 b. An electric field of 455 V/cm wasapplied from well 95 a to well 95 b.

The electric field forces the positively-charged dye towards well 95 b(the negative electrode). The polarity was then switched to confirm thatthe dye would change direction towards well 95 a.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-23. (canceled)
 24. The method according to claim 36, wherein saidlabeled nucleotide triphosphate (NTP) having a detectable moiety is aNTP having a γ-phosphate with a detectable moiety attached thereto. 25.The method according to claim 36, wherein said charged detectable moietywhen released comprises a pyrophosphate with a fluorophore moietyattached thereto.
 26. The method according to claim 24, wherein saidlabeled NTP is incorporated into said nucleic acid primer hybridized tosaid target nucleic acid using said polymerase, thereby releasing saidγ-phosphate with said detectable moiety attached thereto.
 27. The methodaccording to claim 26, wherein said target nucleic acid comprises aself-complementary region forming said primer.
 28. The method accordingto claim 35, wherein the charge of said detectable moiety after releaseis different than said labeled nucleotide phosphate (NP) having adetectable moiety attached thereto.
 29. The method according to claim28, wherein the charge of said detectable moiety is more positive thanthe unincorporated labeled NP.
 30. The method according to claim 28,wherein the charge of said detectable moiety attached thereto isopposite in sign compared to the unincorporated fluorescently labeledNP.
 31. The method according to claim 35, further comprising c)measuring said detectable moiety with a measuring device.
 32. The methodaccording to claim 31, wherein said measuring device is selected fromthe group consisting of a charge coupled device (CCD) camera, aphotodiode, a video chip, amp meter, voltage meter, and adye-impregnated polymeric coating on optical fiber sensor.
 33. Themethod according to claim 32, wherein said detection is via a CCDcamera.
 34. The method according to claim 32, wherein said detection isvia a photodiode.
 35. An analytical method for separating an intact NPprobe from a phosphate detectable moiety, said method comprising: a)providing a sample comprising an intact NP probe with a detectablemoiety attached thereto, whereupon enzymatic cleavage of said intact NPprobe, which produces a phosphate detectable moiety, said phosphatedetectable moiety carries a molecular charge which is different than themolecular charge of said intact NP probe; and b) applying an energyfield to said sample, thereby separating said phosphate detectablemoiety from said intact NP probe.
 36. The method according to claim 35,wherein said NP probe with a detectable moiety is a labeled nucleotidetriphosphate (NTP). 37.-50. (canceled)